Longevity Wiki - User contributions [en-GB]

Protein restriction

2022-11-30T19:39:55Z

Rapacurious: /* Mice */ added mice lifespan references

Reduced intake of energy ([[calorie restriction]]) has a long history in longevity research, but for almost as long scientists have been interested in understanding if restriction of specific types of macronutrients would recapitulate the effects of CR. The results of many early studies were mixed, likely due to differences in dietary protein quality and the degree of restriction.<ref name=":2">Green CL, Lamming DW. Regulation of metabolic health by essential dietary amino acids. ''Mech Ageing Dev.'' 2019 Jan;177:186-200. doi:[https://doi.org/10.1016%2Fj.mad.2018.07.004 10.1016/j.mad.2018.07.004]. [Epub 2018 Jul 22]. PMID: 30044947; PMCID: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333505/ PMC6333505]</ref> However, since the end of the last century, interest in protein restriction (PR) as an intervention has been rekindled by studies which have shown that in flies and mice, total protein restriction or restriction of specific essential amino acids can extend lifespan independently of calorie intake.<ref>Mair, W., Piper, M., & Partridge, L. (2005). Calories Do Not Explain Extension of Life Span by Dietary Restriction in Drosophila. ''Plos Biology'', ''3''(7), e223. doi: 10.1371/journal.pbio.0030223</ref><ref name=":9">Solon-Biet SM, McMahon AC, Ballard JW, Ruohonen K, Wu LE, Cogger VC, Warren A, Huang X, Pichaud N, Melvin RG, Gokarn R, Khalil M, Turner N, Cooney GJ, Sinclair DA, Raubenheimer D, Le Couteur DG, Simpson SJ. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab. 2014 Mar 4; 19(3):418-30. doi: [https://doi.org/10.1016/j.cmet.2014.02.009 10.1016/j.cmet.2014.02.009]. </ref>

== Biological mechanism ==
[[File:Graphical abstract for "Regulation of metabolic health by essential dietary amino acids" (Green and Lamming, 2019)..jpg|thumb|Graphical abstract for "Regulation of metabolic health by essential dietary amino acids" (Green and Lamming, 2019).<ref>Green, C., & Lamming, D. (2019). Regulation of metabolic health by essential dietary amino acids. ''Mechanisms Of Ageing And Development'', ''177'', 186-200. doi: 10.1016/j.mad.2018.07.004</ref>]]
Dietary conditions of protein restriction lead to a significant increase in circulating levels of fibroblast growth factor 21 (FGF21), an insulin-sensitizing hormone. Studies in mice and rats show that circulating FGF21 is increased 10-fold within 24 hours of PR and causes subsequent activation of eukaryotic initiation factor 2α (eIF2α) in the liver through the general control nonderepresssible 2 (GNC2) kinase.<ref name=":1">Laeger, T., Henagan, T., Albarado, D., Redman, L., Bray, G., Noland, R., Münzberg, H., Hutson, S., Gettys, T., Schwartz, M. and Morrison, C., 2014. FGF21 is an endocrine signal of protein restriction. ''Journal of Clinical Investigation'', 124(9), pp.3913-3922.</ref> FGF21 appears responsible for the metabolic remodelling associated to PR, namely reduced body weight, energy expenditure and altered food intake, as these outcomes are not observed in FGF21-deficient animals subjected to PR.<ref name=":1" /> The transcription factor ATF4 is required to upregulate FGF21 and other genes necessary to respond to PR and amino acid restriction.<ref>De Sousa-Coelho, A., Marrero, P., & Haro, D. (2012). Activating transcription factor 4-dependent induction of FGF21 during amino acid deprivation. ''Biochemical Journal'', ''443''(1), 165-171. doi: 10.1042/bj20111748</ref>

Interestingly, increases in the circulating levels of FGF21 are not observed in PR diets with reduced BCAAs, suggesting this form of PR might function via a metabolically distinct pathway.<ref name=":0" /> Branched-chain amino acids or BCAAs (leucine, isoleucine and valine) are among the nine essential amino acids for humans and have an aliphatic side-chain with a branch. BCAA dietary restriction extends lifespan in fruit flies and mice by modulating the mTOR signalling pathway, the target of [[rapamycin]].<ref>Hill, C. and Kaeberlein, M., 2021. Anti-ageing effects of protein restriction unpacked. ''Nature'', 589(7842), pp.357-358.</ref> Sestrin, an inhibitor of mTOR complex 1 (mTORC1), has recently been implicated as a sensor of amino acids in the intestine, and has been shown to maintain homeostasis and regulate lifespan in flies.<ref>Lu, J., Temp, U., Müller-Hartmann, A., Esser, J., Grönke, S. and Partridge, L., 2020. Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids. ''Nature Aging'', 1(1), pp.60-72.</ref>

Restricting methionine, another essential amino acid in humans, by approximately 70-80% has also been shown to be sufficient to increase lifespan in mice and to provide some of the health benefits associated to [[Calorie restriction|caloric restriction]] (CR).<ref>Miller, R., Buehner, G., Chang, Y., Harper, J., Sigler, R., & Smith-Wheelock, M. (2005). Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. ''Aging Cell'', ''4''(3), 119-125. doi: 10.1111/j.1474-9726.2005.00152.x</ref><ref>Forney, L., Wanders, D., Stone, K., Pierse, A., & Gettys, T. (2017). Concentration-dependent linkage of dietary methionine restriction to the components of its metabolic phenotype. ''Obesity'', ''25''(4), 730-738. doi: 10.1002/oby.21806</ref> Methionine restriction is hypothesized to be mediated via FGF21 and to inhibit mTORC1, although this might occur in a sexually dimorphic way and be exclusive to male mice.<ref>Nichenametla, S., Mattocks, D., Malloy, V., & Pinto, J. (2018). Sulfur amino acid restriction-induced changes in redox-sensitive proteins are associated with slow protein synthesis rates. ''Annals Of The New York Academy Of Sciences'', ''1418''(1), 80-94. doi: 10.1111/nyas.13556</ref><ref>Douris, N., Stevanovic, D., Fisher, f., Cisu, T., Chee, M., & Nguyen, N. et al. (2015). Central Fibroblast Growth Factor 21 Browns White Fat via Sympathetic Action in Male Mice. ''Endocrinology'', ''156''(7), 2470-2481. doi: 10.1210/en.2014-2001</ref><ref>Yu, D., Yang, S., Miller, B., Wisinski, J., Sherman, D., & Brinkman, J. et al. (2018). Short‐term methionine deprivation improves metabolic health via sexually dimorphic, mTORCI‐independent mechanisms. ''The FASEB Journal'', ''32''(6), 3471-3482. doi: 10.1096/fj.201701211r</ref>

The relationship between amino acid intake and metabolic health is complex and no clear consensus exists yet as to the exact mechanism through which benefit might be derived, as well as its effect in other less studied tissues such as the brain.

== Human studies ==
A number of long-term studies suggest that lower protein diets in humans are associated to improved metabolic health and increased lifespan.

==== NHANES III ====
A restrospective cohort study based in the National Health And Nutrition Examination Survey (NHANES) III, surveyed 6,381 men and women located in the USA and of 50 years old and over. They found that participants of 50 to 65 years old with high animal-derived protein intake had a 75% increased risk in mortality and a 4-fold increase in risk of age-associated diseases such as cancer.<ref>Levine, M., Suarez, J., Brandhorst, S., Balasubramanian, P., Cheng, C., & Madia, F. et al. (2014). Low Protein Intake Is Associated with a Major Reduction in IGF-1, Cancer, and Overall Mortality in the 65 and Younger but Not Older Population. ''Cell Metabolism'', ''19''(3), 407-417. doi: 10.1016/j.cmet.2014.02.006</ref> However, the reverse trend was observed in participants aged over 65 years old, in which high animal-derived protein intake was associated to lower cancer risk and mortality. Independently of age, high protein intake was associated to a 5-fold increase in the risk of diabetes, although it is important to observe that the particular sample size for diabetes-related deaths was low (n=21). Of note, this study controlled for age, sex, waist circumference and total calories consumption, but did not control for amount of exercise.

Interestingly, the relationships between protein intake, health and mortality risk were no longer observed if protein consumed was plant-derived. When controlling for plant-derived protein, no positive nor negative associations were observed, suggesting that plant-derived protein has no added health benefits but rather that animal-derived protein has deleterious health effects. These findings are in agreement with previous studies showing consumption of red meat is associated to higher risk of all-cause mortality and cancer.<ref>Sun, Q. (2012). Red Meat Consumption and Mortality. ''Archives Of Internal Medicine'', ''172''(7), 555. doi: 10.1001/archinternmed.2011.2287</ref>

This study also measured levels of insulin-like growth factor 1 (IGF-1) in a subset of the cohort (n= 2,253 subjects) and found that low protein intake was associated with a significant reduction in IGF-1 and in turn with a lower incidence of cancer. Higher circulating levels of IGF-1, the molecule that negatively regulates the longevity gene [[FOXO longevity genes|FOXO]], has been extensively associated with an increased risk of cancer and might act as a promoter of tumorigenesis.<ref>Grimberg, A. (2003). Mechanisms by which IGF-I May Promote Cancer. ''Cancer Biology &Amp; Therapy'', ''2''(6), 628-633. doi: 10.4161/cbt.2.6.678</ref><ref>Hua, H., Kong, Q., Yin, J., Zhang, J., & Jiang, Y. (2020). Insulin-like growth factor receptor signaling in tumorigenesis and drug resistance: a challenge for cancer therapy. ''Journal Of Hematology &Amp; Oncology'', ''13''(1). doi: 10.1186/s13045-020-00904-3</ref><ref>Pollak, M., Schernhammer, E., & Hankinson, S. (2004). Insulin-like growth factors and neoplasia. ''Nature Reviews Cancer'', ''4''(7), 505-518. doi: 10.1038/nrc1387</ref>

==== (EPIC)-NL ====
A large prospective cohort study based on the European Prospective Investigation into Cancer and Nutrition (EPIC)-NL study, measured dietary protein intake and incidence of diabetes among 38,094 participants in Europe.<ref>Sluijs, I., Beulens, J., van der A, D., Spijkerman, A., Grobbee, D., & van der Schouw, Y. (2009). Dietary Intake of Total, Animal, and Vegetable Protein and Risk of Type 2 Diabetes in the European Prospective Investigation into Cancer and Nutrition (EPIC)-NL Study. ''Diabetes Care'', ''33''(1), 43-48. doi: 10.2337/dc09-1321</ref> Similarly to the NHANES III study, they found that higher animal protein intake was associated with increased risk of diabetes. However, in discrepancy to the NHANES III study, the (EPIC)-NL also found an association, although more nuanced, between higher total protein intake and increased incidence of diabetes which was independent of protein source.

==== RCTs ====
A small randomised controlled trial (RCT) showed that moderate protein restriction (PR) improved several health markers in both humans and mice.<ref name=":0">Fontana, L., Cummings, N., Arriola Apelo, S., Neuman, J., Kasza, I., & Schmidt, B. et al. (2016). Decreased Consumption of Branched-Chain Amino Acids Improves Metabolic Health. ''Cell Reports'', ''16''(2), 520-530. doi: 10.1016/j.celrep.2016.05.092</ref> They observed that 7-9% PR led to improved blood glucose levels and blood glucose tolerance, while decreasing body mass index (BMI) and fat mass in both humans and mice. However, the human subjects of the study (n=38) were overweight or mildly obese, and the control group might have been consuming an excessive amount of protein that consisted of more than 50% of their diet. On the contrary, mice in the control group were fed a diet that consisted of 21% protein.

In the same study, they fed C57BL/6J wild-type mice a diet with reduced branched-chain amino acids (BCAAs) and found it was sufficient to improve metabolic health markers and body composition and had equivalent health effects to those of total PR.

== Animal studies ==
Studies in rodents on the effect of PR consistently show that reduced protein intake, independently of total calorie consumption, leads to a variety of improved metabolic health parameters, as well as increased food intake and energy expenditure.<ref name=":2" /> Some scientists argue that PR has wider health benefits over traditional [[Calorie restriction|CR]], which derive from the additional energy expenditure observed in PR diets.<ref name=":3">Laeger, T., Henagan, T., Albarado, D., Redman, L., Bray, G., & Noland, R. et al. (2014). FGF21 is an endocrine signal of protein restriction. ''Journal Of Clinical Investigation'', ''124''(9), 3913-3922. doi: 10.1172/jci74915</ref><ref name=":4">Morrison, C., Xi, X., White, C., Ye, J., & Martin, R. (2007). Amino acids inhibit Agrp gene expression via an mTOR-dependent mechanism. ''American Journal Of Physiology-Endocrinology And Metabolism'', ''293''(1), E165-E171. doi: 10.1152/ajpendo.00675.2006</ref><ref name=":5">White, P., Lapworth, A., An, J., Wang, L., McGarrah, R., & Stevens, R. et al. (2016). Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export. ''Molecular Metabolism'', ''5''(7), 538-551. doi: 10.1016/j.molmet.2016.04.006</ref>

==== Mice ====
In C57BL/6 mice of both sexes fed ad libitum, 5% dietary protein during 14 months led to increased food intake, increased adiposity, improved gluocose tolerance, reduced circulating IGF-1 and reduced mTOR activation in comparison to mice fed 60% protein.<ref name=":6">Solon-Biet, S., McMahon, A., Ballard, J., Ruohonen, K., Wu, L., & Cogger, V. et al. (2014). The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice. ''Cell Metabolism'', ''19''(3), 418-430. doi: 10.1016/j.cmet.2014.02.009</ref> Researchers argued that the benefits of [[Calorie restriction|caloric restriction]] are maximized when controlling for the ratio of macronutrients rather than total caloric intake.<ref name=":6" /> Another study looked at male C57BL/6 mice fed 5% protein during a timeframe of only 14 days, and found that besides increased food intake and energy expenditure, there were increased levels of circulating FGF21 and increased expression of eIF2α in the liver, supporting the current understanding of the underlying biological mechanism.<ref name=":3" />

A range of other PR studies in C57BL/6 mice ranging from 4-7% of total protein in diet during intervals of 12-27 weeks showed similar positive outcomes and, additionally, recorded weight loss, reduced fat mass gain, reduced lean mass, improved glucose and pyruvate tolerance and improved expression of health markers in BAT (brown adipose tissue) and WAT (white adipose tissue).<ref name=":0" /><ref>Laeger, T., Albarado, D., Burke, S., Trosclair, L., Hedgepeth, J., & Berthoud, H. et al. (2016). Metabolic Responses to Dietary Protein Restriction Require an Increase in FGF21 that Is Delayed by the Absence of GCN2. ''Cell Reports'', ''16''(3), 707-716. doi: 10.1016/j.celrep.2016.06.044</ref><ref>Maida, A., Zota, A., Sjøberg, K., Schumacher, J., Sijmonsma, T., & Pfenninger, A. et al. (2016). A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution. ''Journal Of Clinical Investigation'', ''126''(9), 3263-3278. doi: 10.1172/jci85946</ref><ref>Solon-Biet, S., Cogger, V., Pulpitel, T., Heblinski, M., Wahl, D., & McMahon, A. et al. (2016). Defining the Nutritional and Metabolic Context of FGF21 Using the Geometric Framework. ''Cell Metabolism'', ''24''(4), 555-565. doi: 10.1016/j.cmet.2016.09.001</ref><ref>Hill, C., Laeger, T., Albarado, D., McDougal, D., Berthoud, H., Münzberg, H., & Morrison, C. (2017). Low protein-induced increases in FGF21 drive UCP1-dependent metabolic but not thermoregulatory endpoints. ''Scientific Reports'', ''7''(1). doi: 10.1038/s41598-017-07498-w</ref><ref>Cummings, N., Williams, E., Kasza, I., Konon, E., Schaid, M., & Schmidt, B. et al. (2017). Restoration of metabolic health by decreased consumption of branched-chain amino acids. ''The Journal Of Physiology'', ''596''(4), 623-645. doi: 10.1113/jp275075</ref>

Several different studies have found that PR in the 5-7% range increases the lifespan of C57BL/6J male mice <ref name=":9" /><ref>Richardson NE, Konon EN, Schuster HS, Mitchell AT, Boyle C, Rodgers AC, Finke M, Haider LR, Yu D, Flores V, Pak HH, Ahmad S, Ahmed S, Radcliff A, Wu J, Williams EM, Abdi L, Sherman DS, Hacker T, Lamming DW. Lifelong restriction of dietary branched-chain amino acids has sex-specific benefits for frailty and lifespan in mice. Nature Aging, 2021 Jan;1(1):73-86. doi: 10.1038/s43587-020-00006-2. Epub 2021 Jan 14. PMID: 33796866 PMCID: [http://www.ncbi.nlm.nih.gov/pmc/articles/pmc8009080/ PMC8009080].

</ref>, and this effect has been shown to require FGF21.<ref>Hill CM, Albarado DC, Coco LG, Spann RA, Khan MS, Qualls-Creekmore E, Burk DH, Burke SJ, Collier JJ, Yu S, McDougal DH, Berthoud HR, Münzberg H, Bartke A, Morrison CD. FGF21 is required for protein restriction to extend lifespan and improve metabolic health in male mice. Nature Communications, 2022 Apr 7;13(1):1897. doi: 10.1038/s41467-022-29499-8. PMID: 35393401. PMCID: [http://www.ncbi.nlm.nih.gov/pmc/articles/pmc8991228/ PMC8991228].

</ref>

==== Rats ====

Sprague-Dawley male rats fed ad libitum with 9% of protein in diets during 14 days, led to increased food intake and increased levels of FGF21 and eIF2α in the liver, similarly to mice.<ref name=":3" /> In another study of Sprague-Dawley male rats with 10% dietary protein intake during 14 days, increased hepatic [[autophagy]] and reduced hepatic lipogenic expression was observed.<ref>Henagan, T., Laeger, T., Navard, A., Albarado, D., Noland, R., & Stadler, K. et al. (2016). Hepatic autophagy contributes to the metabolic response to dietary protein restriction. ''Metabolism'', ''65''(6), 805-815. doi: 10.1016/j.metabol.2016.02.015</ref>

However, obesity-prone rats with completely abolished protein intake (0%), showed decreased energy intake and induced fatty liver which persisted after the restriction period, which can be associated to poor health.<ref name=":7">Pezeshki, A., Zapata, R., Singh, A., Yee, N., & Chelikani, P. (2016). Low protein diets produce divergent effects on energy balance. ''Scientific Reports'', ''6''(1). doi: 10.1038/srep25145</ref> In the same study, energy intake was increased and there was no incidence of fatty liver in rats that were fed 5% protein compared to 15% protein in the control diet.<ref name=":7" /> This study shows that moderate, but not complete, protein restriction might be beneficial to health.

Additionally, in Wistar fatty rats, an animal model of type 2 diabetes and obesity, low-protein diets demonstrated a health benefit in diabetic status and prevented diabetic nephropathy.<ref>Kitada, M., Ogura, Y., Suzuki, T., Monno, I., Kanasaki, K., Watanabe, A., & Koya, D. (2018). A low-protein diet exerts a beneficial effect on diabetic status and prevents diabetic nephropathy in Wistar fatty rats, an animal model of type 2 diabetes and obesity. ''Nutrition &Amp; Metabolism'', ''15''(1). doi: 10.1186/s12986-018-0255-1</ref>

== Caveats ==
Together with a range of health benefits, [[Calorie restriction|caloric restriction]] (CR) is often accompanied by a number of important caveats.<ref>Ingram, D., & de Cabo, R. (2017). Calorie restriction in rodents: Caveats to consider. ''Ageing Research Reviews'', ''39'', 15-28. doi: 10.1016/j.arr.2017.05.008</ref> Some of the deleterious effects of CR include reduced bone density,<ref>Baek, K., Barlow, A., Allen, M., & Bloomfield, S. (2008). Food restriction and simulated microgravity: effects on bone and serum leptin. ''Journal Of Applied Physiology'', ''104''(4), 1086-1093. doi: 10.1152/japplphysiol.01209.2007</ref> impaired wound healing<ref>Hunt, N., Li, G., Zhu, M., Levette, A., Chachich, M., & Spangler, E. et al. (2011). Effect of calorie restriction and refeeding on skin wound healing in the rat. ''AGE'', ''34''(6), 1453-1458. doi: 10.1007/s11357-011-9321-6</ref> and an increased risk of infection.<ref>Kristan, D. (2008). Calorie restriction and susceptibility to intact pathogens. ''AGE'', ''30''(2-3), 147-156. doi: 10.1007/s11357-008-9056-1</ref>

Similarly, recent studies have shown that protein restriction (PR) can increase frailty and shorten the lifespan of animals when PR is performed under stressful conditions.<ref name=":8">Zajitschek, Felix, et al. “Dietary Restriction Fails to Extend Life in Stressful Environments.” 2022, <nowiki>https://doi.org/10.1101/2022.10.17.512576</nowiki>. </ref> Moderate protein restriction in ''Drosophila'' fruit flies led to lifespan extension at benign temperatures of 25-27ºC, but there was no effect or even a shortening of lifespan in cold (21-23ºC) or hot (29ºC) temperatures.<ref name=":8" /><references />
[[Category:Draft]]
[[Category:Longevity]]

Rapamycin

2022-08-14T15:54:28Z

Rapacurious: /* Safety */ added more references

[[File:Rapamycin2.jpg|thumb|211x211px|The chemical structure of rapamycin.]]
Rapamycin, also known by its brand name Rapamune®, is a compound used to prevent the rejection of organ transplants by the immune system. Rapamycin is a natural antifungal produced by soil bacteria of Eastern Island, named after its native island, Rapa Nui.<ref>''Sirolimus - Wikipedia''. En.wikipedia.org. (2021). Retrieved 27 May 2021, from <nowiki>https://en.wikipedia.org/wiki/Sirolimus</nowiki>.</ref>

Rapamycin was approved by the US Food and Drug Administration in September 1999 and is marketed under the trade name Rapamune® by Pfizer.<ref>Accessdata.fda.gov. 2021. ''Drug Approval Package: Rapamune (Sirolimus) NDA# 021083''. [online] Available at: <<nowiki>https://www.accessdata.fda.gov/drugsatfda_docs/nda/99/21083A.cfm</nowiki>> [Accessed 27 May 2021].</ref> At a high dose, rapamycin has an immunosuppressant function that is used in preventing rejection of kidney transplants by the immune system. It is also used to coat coronary stents, and to treat rare lung diseases.<ref>Doggrell, S. (2006). Sirolimus- or paclitaxel-eluting stents for coronary artery revascularisation. ''Expert Opinion On Pharmacotherapy'', ''7''(2), 225-228. <nowiki>https://doi.org/10.1517/14656566.7.2.225</nowiki></ref> The patent on rapamycin has expired, and pharmacological companies have developed similar drugs such as everolimus.<ref>Cancer, C. (2021). ''Everolimus - Drug Information - Chemocare''. Chemocare.com. Retrieved 27 May 2021, from <nowiki>http://chemocare.com/chemotherapy/drug-info/everolimus.aspx</nowiki>.</ref>

Rapamycin has been shown to extend healthy lifespan in worms, yeast, flies, and mice.<ref>[https://science.sciencemag.org/content/328/5976/321.abstract?casa_token=ptqabKjuDxMAAAAA:qBvrJQ01NpHQ8WyJSQf6HeQIhaaexgZXZMvi9i_AP4fE2iHhvMzLV-z3_eF5T3rEauJkg0eQNQFfbA Fontana, L., Partridge, L., & Longo, V. D. (2010). Extending healthy life span—from yeast to humans. ''science'', ''328''(5976), 321-326.]</ref> Some physicians and scientists have thus suggested that rapamycin may slow down aging.<ref name=":2" /><ref>Johnson, S. C., Rabinovitch, P. S., & Kaeberlein, M. (2013). mTOR is a key modulator of ageing and age-related disease. ''Nature'', ''493''(7432), 338-345.</ref>

Scientists who study the biology of aging believe that, due to evidence of rapamycin slowing aging in animals, it could be used to prevent the onset of all age-related diseases in humans – to become one of the first identified “anti-aging drugs”.<ref name=":2">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814615/ Blagosklonny, M. (2019). Rapamycin for longevity: opinion article. ''Aging'', ''11''(19), 8048-8067. https://doi.org/10.18632/aging.102355]</ref><ref>Blagosklonny, M. (2006). Aging and Immortality: Quasi-Programmed Senescence and Its Pharmacologic Inhibition. ''Cell Cycle'', ''5''(18), 2087-2102. <nowiki>https://doi.org/10.4161/cc.5.18.3288</nowiki></ref> It is being repurposed for humans as an anti-aging drug, such as in the PEARL clinical study, which is expected to conclude in 2023.<ref>[https://clinicaltrials.gov/ct2/show/NCT04488601 ''Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity Study - Full Text View - ClinicalTrials.gov''. Clinicaltrials.gov. (2021). Retrieved 27 May 2021, from https://clinicaltrials.gov/ct2/show/NCT04488601.]</ref>

== Evidence of increased healthspan or lifespan ==

=== Dogs ===
There is preliminary evidence that rapamycin may prevent age-related decline in dogs. One study showed statistically significant improvements in heart function in dogs receiving rapamycin, relative to those that received placebo, similar to what has been observed in older laboratory mice.<ref name=":9">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411365/ Urfer, S. R., Kaeberlein, T. L., Mailheau, S., Bergman, P. J., Creevy, K. E., Promislow, D. E., & Kaeberlein, M. (2017). A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs. ''Geroscience'', ''39''(2), 117-127.]</ref> As part of the Dog Aging Project at the University of Washington, the TRIAD study is testing whether rapamycin can extend healthy lifespan in pet dogs.
=== Mice ===
In multiple studies in different breeds of mice, rapamycin demonstrates a robust effect on increasing healthy lifespan. Rapamycin significantly extends lifespan in approximately 90% of the mice models it has been tested in.<ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.</ref>

In 2009, rapamycin was shown to increase the lifespan of both male and female mice when given in late life (600 days).<ref name=":5">[https://doi.org/10.1038%2Fnature08221 Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., ... & Miller, R. A. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. ''nature'', ''460''(7253), 392-395.]</ref> Mean survival was extended by 28% for males and 38% for females, while maximal lifespan increased by 9% for males and 14% for females.<ref name=":5" /> This was the first evidence that the lifespan of a mammal could be significantly increased by a pharmacological drug. This mouse study is special because the results were obtained following the US National Institute on Aging's Interventions Testing Program (ITP) protocol. The ITP is regarded as the gold standard for testing drugs that target aging.<ref>Nadon, N. L., Strong, R., Miller, R. A., Nelson, J., Javors, M., Sharp, Z. D., ... & Harrison, D. E. (2008). Design of aging intervention studies: the NIA interventions testing program. ''Age'', ''30''(4), 187-199.</ref>

The landmark 2009 study also showed that rapamycin could increase healthy lifespan when given in old age. This has important implications for human testing, as it suggests that the drug might still exhibit healthspan and lifespan benefits even when given to the elderly. Rapamycin contrasts with calorie restriction in this regard; some evidence suggests that calorie restriction needs to be practiced from early adulthood, and may even fail to provide benefit for animals that are already old.<ref>Szafranski, K., & Mekhail, K. (2014). The fine line between lifespan extension and shortening in response to caloric restriction. ''Nucleus'', ''5''(1), 56-65.</ref>
==== Rapidly aging mice models ====
Using a mouse model that mimics the accelerated aging disease Hutchinson-Gilford progeria, rapamycin was shown to increase lifespan by over 50%. It also improved cardiac and skeletal muscle function in these mice.<ref>[https://doi.org/10.1126%2Fscitranslmed.3003802 Ramos, F. J., Chen, S. C., Garelick, M. G., Dai, D. F., Liao, C. Y., Schreiber, K. H., ... & Kennedy, B. K. (2012). Rapamycin reverses elevated mTORC1 signaling in lamin A/C–deficient mice, rescues cardiac and skeletal muscle function, and extends survival. ''Science translational medicine'', ''4''(144), 144ra103-144ra103.]
</ref> In one short-lived mutant strain of mice that mimics Leigh syndrome, rapamycin was shown to extend maximum life span nearly three-fold.<ref>[https://www.ncbi.nlm.nih.gov/pubmed/24231806/ Johnson, S. C., Yanos, M. E., Kayser, E. B., Quintana, A., Sangesland, M., Castanza, A., ... & Kaeberlein, M. (2013). mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome. ''Science'', ''342''(6165), 1524-1528.]</ref>

==== Middle-aged mice ====
Several recent studies have shown that rapamycin can extend the lifespan of middle-aged or aged mice. One study showed that treating 20-month-old mice (the equivalent of 56–69 years in humans) with rapamycin for only 3 months resulted in a dramatic increase in median lifespan of up to 60%.<ref>Bitto, A., Ito, T.K., Pineda, V.V., LeTexier, N.J., Huang, H.Z., Sutlief, E., Tung, H., Vizzini, N., Chen, B., Smith, K. and Meza, D., 2016. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. ''elife'', ''5'', p.e16351.</ref> A study from 2020 showed that administering rapamycin in late life enhanced the lifespan of male but not female mice, providing evidence for sex differences in rapamycin response.<ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13269 Strong, R., Miller, R. A., Bogue, M., Fernandez, E., Javors, M. A., Libert, S., ... & Harrison, D. E. (2020). Rapamycin‐mediated mouse lifespan extension: Late‐life dosage regimes with sex‐specific effects. ''Aging cell'', ''19''(11), e13269.]</ref> Aged female mice administered rapamycin once every 5 days starting at 20 months of age also extended lifespan.<ref>Arriola Apelo SI, Pumper CP, Baar EL, Cummings NE, Lamming DW. Intermittent administration of rapamycin extends the lifespan of female C57BL/6J mice. ''J Gerontol A Biol Sci Med Sci''. 2016 Jul; 71(7):876-81. doi: [https://academic.oup.com/biomedgerontology/article/71/7/876/2605199 10.1093/gerona/glw064]. Epub 2016 Apr 18. PMID: 27091134; PMCID: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906329/ PMC4906329]</ref> These studies were important as they suggest that much of the health and longevity benefits of rapamycin could be achieved even when dosed in late life or intermittently, as opposed to only being effective with continual dosing in early life.

=== Yeast ===
Inhibition of TOR signalling by rapamycin significantly increases the lifespan of yeast known as ''Saccharomyces cerevisiae.''<ref>Powers, R. W., Kaeberlein, M., Caldwell, S. D., Kennedy, B. K., & Fields, S. (2006). Extension of chronological life span in yeast by decreased TOR pathway signaling. ''Genes & development'', ''20''(2), 174-184.</ref>

=== Flies ===
Rapamycin extends the lifespan of the fruitfly, ''Drosophila melanogaster.'' The extent of lifespan extension observed is beyond what is achievable by flies undergoing other pro-longevity interventions like dietary restriction, or in mutant flies with mild decrements in insulin/insulin-like growth factor signaling (IIS).<ref>Bjedov, I., Toivonen, J. M., Kerr, F., Slack, C., Jacobson, J., Foley, A., & Partridge, L. (2010). Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. ''Cell metabolism'', ''11''(1), 35-46.</ref> Combining rapamycin with two other drugs that target metabolic pathways, lithium and trametinib, results in additive lifespan extension effects, substantially increasing ''Drosophila'' lifepsan by 48%.<ref>Castillo-Quan, J. I., Tain, L. S., Kinghorn, K. J., Li, L., Grönke, S., Hinze, Y., ... & Partridge, L. (2019). A triple drug combination targeting components of the nutrient-sensing network maximizes longevity. ''Proceedings of the National Academy of Sciences'', ''116''(42), 20817-20819.</ref>

=== Roundworms ===
TOR inhibition by rapamycin extends lifespan in ''Caenorhabditis elegans,'' a roundworm nematode widely used in research areas of the biology of aging. The beneficial effects of rapamycin in ''C. elegans'' seem to be mediated via the SKN-1/Nrf and DAF-16/FoxO pathways.<ref>Robida-Stubbs, S., Glover-Cutter, K., Lamming, D. W., Mizunuma, M., Narasimhan, S. D., Neumann-Haefelin, E., ... & Blackwell, T. K. (2012). TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. ''Cell metabolism'', ''15''(5), 713-724.</ref>

=== Age-related diseases ===
Rapamycin has been investigated in specific diseases, showing major impacts on reducing mouse cancer risk, cardiac diseases, neurodegenerative-like processes, and many other pathologies.<ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.
</ref>

===== Cancer =====
In the transgenic HER-2/neu mouse model, mice die prematurely due to susceptibility to cancer.<ref name=":6" /> Rapamycin was hypothesized to improve survival in this model due to its ability to slow aging, which would also address an age-related disease like cancer.<ref name=":6" /> The drug was shown to extend maximal lifespan, by delaying aging in multiple different organs and also suppressing cancer development.<ref name=":6">Anisimov, V. N., Zabezhinski, M. A., Popovich, I. G., Piskunova, T. S., Semenchenko, A. V., Tyndyk, M. L., ... & Blagosklonny, M. V. (2010). Rapamycin extends maximal lifespan in cancer-prone mice. ''The American journal of pathology'', ''176''(5), 2092-2097.</ref>

Other studies suggest that rapamycin can extend lifespan in mouse models where cancer naturally develops, such as in mice prone to cancer due to the Apc tumor suppressor gene mutation, or in mice heterozygous for the Rb1 tumor supressor gene, among others.<ref>Hasty, P., Livi, C. B., Dodds, S. G., Jones, D., Strong, R., Javors, M., ... & Sharp, Z. D. (2014). eRapa restores a normal life span in a FAP mouse model. ''Cancer Prevention Research'', ''7''(1), 169-178.</ref><ref>Livi, C. B., Hardman, R. L., Christy, B. A., Dodds, S. G., Jones, D., Williams, C., ... & Sharp, Z. D. (2013). Rapamycin extends life span of Rb1+/− mice by inhibiting neuroendocrine tumors. ''Aging (Albany NY)'', ''5''(2), 100.</ref><ref>Hambright, H. G., Hurez, V., & Curiel, T. J. (2020). Chronic Mechanistic Target of Rapamycin Inhibition: Preventing Cancer to Delay Aging or Vice Versa?. ''Geriatric Oncology'', 111-128.</ref>

A group of investigators in Germany have argued, based on their experiment in C57BL/6J Rj inbred mice, that rapamycin extends lifespan mainly through delaying cancer incidence, instead of via slowed aging.<ref>Neff, F., Flores-Dominguez, D., Ryan, D. P., Horsch, M., Schröder, S., Adler, T., ... & Ehninger, D. (2013). Rapamycin extends murine lifespan but has limited effects on aging. ''The Journal of clinical investigation'', ''123''(8), 3272-3291.</ref> A further analysis of the paper by Johnson et al. pointed to several important limitations of the study, suggesting that such a conclusion may be premature.<ref name=":82">Johnson, S. C., Martin, G. M., Rabinovitch, P. S., & Kaeberlein, M. (2013). Preserving youth: does rapamycin deliver? ''Science translational medicine'', ''5''(211), 211fs40.</ref> Key limitations included the lack of dose-response profiling of rapamycin; studying only the male sex, which is known to respond less to rapamycin likely in part due to sex differences in drug metabolism; lack of reporting on tumor size and incidence, required to determine whether lifespan extension occured only via slowed cancer or from a general effect on aging; and, the cross-sectional nature of the study, which would have reduced sensitivity for detecting age-related organ/tissue changes compared to longitudinal assays.<ref name=":82" /><ref>Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref>Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much? ''Journal of genetics and genomics,'' ''41''(9), 459.</ref>

===== Heart Disease =====
One study investigated the effects of late-life rapamycin dosing in aged female mice, observing a reversal of age-related heart dysfunction.<ref name=":16">Flynn, J. M., O'Leary, M. N., Zambataro, C. A., Academia, E. C., Presley, M. P., Garrett, B. J., ... & Melov, S. (2013). Late‐life rapamycin treatment reverses age‐related heart dysfunction. ''Aging cell'', ''12''(5), 851-862.

</ref> This included benefits to ejection fraction, cardiac hormones, and reduced inflammation, although no effect was observed for heart fibrosis.<ref name=":16" /> Additionally, improvements in behaviour and physical function were demonstrated.<ref name=":16" /> Another study in naturally aged mice showed improvements in cardiac muscle stiffness, diastolic function with rapamycin.<ref name=":7">Quarles, E., Basisty, N., Chiao, Y. A., Merrihew, G., Gu, H., Sweetwyne, M. T., ... & Rabinovitch, P. S. (2020). Rapamycin persistently improves cardiac function in aged, male and female mice, even following cessation of treatment. ''Aging Cell'', ''19''(2), e13086.</ref> Improvements in heart function were shown with only a brief treatment course of 8 weeks.<ref name=":7" /> Benefits persisted even after rapamycin was stopped, which appears consistent with the hypothesis that rapamycin slows aging.<ref name=":7" />

===== Alzheimer's =====
Alzheimer's Disease (AD) is a progressive neurodegenerative disease for which age is the greatest risk factor.<ref>https://www.science.org/doi/10.1126/scitranslmed.aar4289</ref> In an Alzheimer's model of transgenic PDAPP mice, rapamycin was shown to reduce Amyloid-β, one of the hallmarks of AD.<ref name=":8" /> This led to alleviation of AD-like symptoms, such as restored cognition and memory.<ref name=":8">Spilman, P., Podlutskaya, N., Hart, M. J., Debnath, J., Gorostiza, O., Bredesen, D., ... & Galvan, V. (2010). Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-β levels in a mouse model of Alzheimer's disease. ''PloS one'', ''5''(4), e9979.</ref> Similarly, another major AD hallmark known as tau was mitigated by rapamycin in a tauopathy mouse model.<ref name=":14">Ozcelik, S., Fraser, G., Castets, P., Schaeffer, V., Skachokova, Z., Breu, K., ... & Winkler, D. T. (2013). Rapamycin attenuates the progression of tau pathology in P301S tau transgenic mice. ''PloS one'', ''8''(5), e62459.</ref> The mechanism of clearance of these proteins was linked to autophagy, with benefits seen regardless of whether it was dosed early for prevention, or in late life as treatment.<ref name=":14" />

=== Dose response ===
Rapamycin has shown a dose-response in which higher doses produce larger lifespan extension effects. UMHET3 mice of diverse genetic background were treated with varying doses of dietary rapamycin at 4.7, 14, or 42 ppm, revealing that those fed with the highest rapamycin dose had the greatest lifespan extension.<ref>Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref name=":0">[[Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much?. Journal of genetics and genomics= Yi chuan xue bao, 41(9), 459.|Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much? ''Journal of genetics and genomics,'' ''41''(9), 459.]]</ref> Sex differences in response to rapamycin have been hypothesized to also be related to the effective dose, due to male/female differences in drug metabolism. The optimal dose for longevity in mice remains to be seen, but determining this dose will require consideration of the side effect profile of rapamycin.<ref name=":0" />

== Mechanism ==

=== Manipulating metabolic pathways - differences to calorie restriction ===
Rapamycin has often been described as a 'calorie restriction (CR) mimetic'.<ref name=":15">Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref name=":4" /> This is in part because CR also inhibits the nutrient-sensing mammalian target of rapamycin (mTOR) pathway.<ref name=":3">Cornu, M., Albert, V., & Hall, M. N. (2013). mTOR in aging, metabolism, and cancer. ''Current opinion in genetics & development'', ''23''(1), 53-62.</ref> mTOR plays key roles in cellular growth in response to amino acids, including effects that inhibit cancer and aging mechanisms.<ref name=":3" /><ref>Johnson, S. C., Rabinovitch, P. S., & Kaeberlein, M. (2013). mTOR is a key modulator of ageing and age-related disease. ''Nature'', ''493''(7432), 338-345.</ref> However, later studies have disentangled the effects of rapamycin from that of caloric restriction, showing that they differ significantly.<ref name=":15" /><ref name=":4">Unnikrishnan, A., Kurup, K., Salmon, A. B., & Richardson, A. (2020). Is rapamycin a dietary restriction mimetic?. ''The Journals of Gerontology: Series A'', ''75''(1), 4-13.</ref> For example, unlike 5 months of CR, rapamycin does not decrease leptin, insulin, IGF-1, or FGF-21 in genetically diverse UM-HET3 mice.<ref name=":15" /> This has important implications for understanding biological aging, including the possibility of using CR and rapalogs in combination therapy to slow aging.<ref name=":4" />

Though distinct from CR, fasting inhibits muscle-specific mTOR signaling with reduced effect in old vs young mice, indicating a poorer autophagy and proteosomal degradation response with age.<ref>White, Z., White, R. B., McMahon, C., Grounds, M. D., & Shavlakadze, T. (2016). High mTORC1 signaling is maintained, while protein degradation pathways are perturbed in old murine skeletal muscles in the fasted state. ''The international journal of biochemistry & cell biology'', ''78'', 10-21.</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/21179166/ Sengupta, S., Peterson, T. R., Laplante, M., Oh, S., & Sabatini, D. M. (2010). mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. ''Nature'', ''468''(7327), 1100-1104.]</ref> However, the ability for rapamycin to inhibit mTOR appears to remain robust throughout life, and significant extension of median and maximal lifespan can be achieved even when treatment is initiated in mid-to-late life.<ref name=":5" /><ref>Blagosklonny, M. V. (2019). Rapamycin for longevity: opinion article. ''Aging (Albany NY)'', ''11''(19), 8048.
</ref> Rapamycin contrasts significantly with CR and fasting, in that the latter could be detrimental when used in late life.<ref>Kemnitz, J. W. (2011). Calorie restriction and aging in nonhuman primates. ''Ilar Journal'', ''52''(1), 66-77.</ref><ref>Goto, S., Takahashi, R., Radak, Z., & Sharma, R. (2007). Beneficial biochemical outcomes of late‐onset dietary restriction in rodents. ''Annals of the New York Academy of Sciences'', ''1100''(1), 431-441.</ref><ref>Kristan, D. M. (2008). Calorie restriction and susceptibility to intact pathogens. ''Age'', ''30''(2), 147-156.</ref> Rapamycin also targets multiple diseases of aging, but seemingly in a segmented, tissue-specific manner.<ref>Neff, F., Flores-Dominguez, D., Ryan, D. P., Horsch, M., Schröder, S., Adler, T., ... & Ehninger, D. (2013). Rapamycin extends murine lifespan but has limited effects on aging. ''The Journal of clinical investigation'', ''123''(8), 3272-3291.</ref>

=== mTORC1 and mTORC2 ===
In non-mammals the mTOR equivalent is known as the target of rapamycin (TOR), first discovered by a team led by Michael Hall in the yeast ''Saccharomyces cerevisiae''.<ref>Thomas, G., & Hall, M. N. (1997). TOR signalling and control of cell growth. ''Current opinion in cell biology'', ''9''(6), 782-787.</ref><ref>Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., & Hall, M. N. (1993). Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. ''Cell'', ''73''(3), 585-596.</ref> mTOR signalling appears to be evolutionarily conserved, and this extends further to include various mammals, such as mice, rats, and dogs.

Rapamycin acts on mTOR, with multiple signaling functions subdivided across two major protein complexes known as mTORC1 and mTORC2.<ref name=":3" /> There is some evidence suggesting that the health and lifespan benefit of rapamycin is more related to inhibition of mTORC1 than mTORC2.<ref name=":19">Lamming, D. W., Ye, L., Katajisto, P., Goncalves, M. D., Saitoh, M., Stevens, D. M., ... & Baur, J. A. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. ''science'', ''335''(6076), 1638-1643.</ref><ref name=":11">Lamming, D. W., Mihaylova, M. M., Katajisto, P., Baar, E. L., Yilmaz, O. H., Hutchins, A., ... & Sabatini, D. M. (2014). Depletion of Rictor, an essential protein component of m TORC 2, decreases male lifespan. ''Aging cell'', ''13''(5), 911-917.</ref> In mice, males exhibit weaker lifespan extension effects from rapamycin than in females. One study suggests that inhibiting mTORC2 explains why the sex difference in the response to mTOR inhibition by rapamycin.<ref name=":11" />

=== Effects on glucoregulatory control ===
A noted issue regarding rapamycin is the disruption of glucose metabolism with chronic dosing, which has been observed in both humans and mice. In mice, this side effect has been shown to be due in part to disruption of mTORC2 in the liver, leading to hepatic insulin resistance.<ref name=":19" /> This effect has previously been shown to be reversible upon stopping the drug in both lean and obese mice.<ref name=":18">Liu, Y., Diaz, V., Fernandez, E., Strong, R., Ye, L., Baur, J. A., ... & Salmon, A. B. (2014). Rapamycin-induced metabolic defects are reversible in both lean and obese mice. ''Aging (Albany NY)'', ''6''(9), 742.</ref> Whether disrupted glucose metabolism is dispensable for the lifespan extending effects of rapamycin remains controversial.<ref>Blagosklonny, M. V. (2012). Once again on rapamycin-induced insulin resistance and longevity: despite of or owing to. ''Aging (Albany NY)'', ''4''(5), 350.</ref><ref>[https://www.nature.com/articles/s41419-019-1822-8 Blagosklonny, M. V. (2019). Fasting and rapamycin: diabetes versus benevolent glucose intolerance. ''Cell death & disease'', ''10''(8), 1-10.]</ref><ref>[https://www.science.org/doi/full/10.1126/science.1215135 Lamming, D. W., Ye, L., Katajisto, P., Goncalves, M. D., Saitoh, M., Stevens, D. M., ... & Baur, J. A. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. ''science'', ''335''(6076), 1638-1643.]</ref><ref>Strong, R., Miller, R. A., Antebi, A., Astle, C. M., Bogue, M., Denzel, M. S., ... & Harrison, D. E. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α‐glucosidase inhibitor or a Nrf2‐inducer. ''Aging cell'', ''15''(5), 872-884.</ref> Rapamycin has previously been shown to increase insulin sensitivity with acute dosing, while decreasing insulin sensitivity with chronic dosing.<ref>Ye, L., Varamini, B., Lamming, D. W., Sabatini, D. M., & Baur, J. A. (2012). Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2. ''Frontiers in genetics'', ''3'', 177.</ref>

=== Reducing the effects of cellular senescence ===
The accumulation of senescent cells is thought to be an important mechanism underlying aging. Rapamycin is regarded as a senomorphic that may inhibit the pro-inflammatory secretory phenotype produced by senescent cells in humans, mice, and rats.<ref>[[Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., ... & Perez, V. I. (2017). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism. Aging cell, 16(3), 564-574.|Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., ... & Perez, V. I. (2017). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism. ''Aging cell'', ''16''(3), 564-574.]]</ref><ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.
</ref> A preliminary study in humans aged 40 years or older showed that topical rapamycin reduced markers of cellular senescence in the skin and improved its physical appearance.<ref>[https://doi.org/10.1007%2Fs11357-019-00113-y Chung, C. L., Lawrence, I., Hoffman, M., Elgindi, D., Nadhan, K., Potnis, M., ... & Sell, C. (2019). Topical rapamycin reduces markers of senescence and aging in human skin: an exploratory, prospective, randomized trial. ''Geroscience'', ''41''(6), 861-869.]
</ref>

== Human clinical trials ==
[[File:Rapamycin.jpg|thumb|235x235px|Part of the rationale of the PEARL study is to determine the optimal dose of rapamycin to potentially slow aging.<ref name=":2" />]]

=== PEARL study ===
Rapamycin is currently being tested for safety and efficacy in a clinical trial called the Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity (PEARL) study. The clinical trial aims to systematically investigate the use of rapamycin to promote healthy longevity, and is expected to conclude in 2023.<ref>[https://clinicaltrials.gov/ct2/show/NCT04488601 ''Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity Study - Full Text View - ClinicalTrials.gov''. Clinicaltrials.gov. (2021). Retrieved 27 May 2021, from https://clinicaltrials.gov/ct2/show/NCT04488601.]</ref>

The study will begin with 200 adults aged 50 years or older who will receive rapamycin for up to one year. The study is being conducted by AgelessRx, a new company dedicated to developing scientifically supported interventions to prevent and treat age-related diseases, in collaboration with the University of California.<ref>[https://www.longevity.technology/agelessrx-and-funding-an-irb-approved-rapamycin-trial/ ''AgelessRx and funding an IRB-approved rapamycin trial - Longevity.Technology''. Longevity.Technology. (2021). Retrieved 27 May 2021, from https://www.longevity.technology/agelessrx-and-funding-an-irb-approved-rapamycin-trial/.]</ref>

The trial aims to obtain clinical data at 6 and 12 months of treatment, such as via testing of blood, body composition DXA, fecal microbiome, immune function, inflammation, skeletal muscle, and epigenetic aging clocks.

== Dog clinical trials ==
The Dog Aging Project is a US Government NIH-funded initiative investigating dog aging.<ref>https://dogagingproject.org/</ref> The project is led by Professor Matt Kaeberlein at the University of Washington.<ref name=":10">Kaeberlein, M., Creevy, K. E., & Promislow, D. E. (2016). The dog aging project: translational geroscience in companion animals. ''Mammalian genome'', ''27''(7), 279-288.</ref>

The Test of Rapamycin In Aging Dogs (TRIAD) study is investigating rapamycin as a treatment to slow aging in dogs. The investigators hope to increase healthy canine lifespan with rapamycin by delaying the onset of age-related diseases like cancer and heart disease.<ref name=":9" /><ref name=":10" />

Aging biology scientists believe that studying dog aging might not only help improve canine healthspan, but also have implications for humans.<ref name=":10" /> Dogs may be a useful animal model because they share the same environment that humans live in, and suffer from similar chronic diseases with aging.<ref>Hoffman, J. M., Creevy, K. E., Franks, A., O'Neill, D. G., & Promislow, D. E. (2018). The companion dog as a model for human aging and mortality. ''Aging Cell'', ''17''(3), e12737.</ref>

===== Heart disease =====
One randomized-controlled trial in 24 middle-aged dogs treated with low-dose rapamycin showed suggestion of partial reversal of age-related heart dysfunction, as measured via echocardiography. The intervention was well-tolerated, with no clinically meaningful adverse events noted with a non-immunosuppressive dose of rapamycin during the 10 week period. This was a small study over a relatively short duration; further testing in larger clinical studies will be necessary to determine whether rapamycin can be used to treat age-related heart disease in dogs.

== Regulatory approval ==
Rapamycin was approved by the US Food and Drug Administration (FDA) in 1999 to prevent organ rejection in liver transplant patients, and has been marketed under the brand name Rapamune.<ref>[https://link.springer.com/article/10.1007/s11357-020-00274-1 Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.]</ref> The patent on rapamycin has expired, and chemically similar compounds called 'rapalogs' are being researched by biotechnology companies.<ref>[https://pubs.acs.org/doi/full/10.1021/acsmedchemlett.9b00215 Abdel-Magid, A. F. (2019). Rapalogs potential as practical alternatives to rapamycin.]
</ref> It is not currently approved for use as an anti-aging medication, due to lack of human clinical data for this purpose.

== Safety ==
Rapamycin has been used to treat millions of patients over several decades since obtaining FDA approval in 1999. It is generally considered safe in humans, but only when used under clinical supervision for specific indications. Rapamycin and its analogs are immunosuppressants, and used as such in the clinic; some rapalogs have received “black-box” FDA warnings due to the risks of infection, as well as the potential risk of cancer due to suppression of tumor immune surveillance.<ref>https://www.pfizermedicalinformation.com/en-us/rapamune/boxed-warning</ref><ref>https://www.pdr.net/drug-summary/Afinitor-everolimus-416.6101</ref>

Various side effects have been reported with the dose of rapamycin used to prevent rejection in organ transplant patients, who are often concurrently treated with multiple other medications.<ref>Webster, A. C., Lee, V. W., Chapman, J. R., & Craig, J. C. (2006). Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials. ''Transplantation'', ''81''(9), 1234-1248.</ref> These include pain, headache, fever, high blood pressure, glucose intolerance, new-onset diabetes, nausea, abdominal pain, constipation, diarrhea, thrombocytopenia, leukopenia, among others. However, side effects are mostly reversible (at least if therapy is rapidly discontinued) and represent worst-case scenarios, particularly because the patients sampled in clinical studies are already severely ill and taking the drug along with other medications.<ref>Bischof, E., Siow, R.C., Zhavoronkov, A. and Kaeberlein, M., 2021. The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19. ''The Lancet Healthy longevity'', ''2''(2), pp.e105-e111.
</ref> In subjects taking high doses of rapamycin and analogs for severe, chronic conditions including tuberous sclerosis complex, an inherited genetic disorder of increased mTOR signaling, or for cancer, the side effects have occasionally led life-threatening adverse events or death.<ref>Trelinska J, Dachowska I, Kotulska K, Fendler W, Jozwiak S, Mlynarski W. Complications of mammalian target of rapamycin inhibitor anticancer treatment among patients with tuberous sclerosis complex are common and occasionally life-threatening. Anti-cancer drugs. 2015;26(4):437-42. PMID: [https://pubmed.ncbi.nlm.nih.gov/25719621/ 25719621] DOI: [https://doi.org/10.1097/cad.0000000000000207 10.1097/CAD.0000000000000207]</ref><ref>Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S, et al. Phase 3 trial of everolimus for metastatic renal cell carcinoma : final results and analysis of prognostic factors. Cancer. 2010; 116(18):4256-65. PMID: [https://pubmed.ncbi.nlm.nih.gov/20549832/ 20549832] DOI: [https://doi.org/10.1002/cncr.25219 10.1002/cncr.25219] </ref>

Case studies of the safety profile of rapamycin in the context of overdosing have suggested that it may have a large margin of safety or a high median lethal dose, but only in the acute setting.<ref>Ceschi, A., Heistermann, E., Gros, S., Reichert, C., Kupferschmidt, H., Banner, N. R., ... & Taegtmeyer, A. B. (2015). Acute sirolimus overdose: a multicenter case series. ''PLoS One'', ''10''(5), e0128033.</ref> The distinction with chronic high dose mTOR inhibition must be made because resultant immunosuppression can lead to susceptibility to infection with fatal consequences. Some preclinical data suggests that the longevity benefits of rapamycin may be retained via intermittent dosing.<ref name=":2" /> In considering the known clinical data about rapamycin's controversial safety at continuous, high doses, some researchers have proposed that rapamycin should dosed intermittently to minimize side effects while sufficiently inhibiting mTOR for an effect on aging.<ref name=":2" />

From a longevity perspective, there is a lack of published clinical data demonstrating the safety of rapamycin in healthy adults. One small randomized pilot study of rapamycin in 25 older adults aged 70-95 taking 1 mg/day of rapamycin reported finding no clinically significant effects, including a lack of effect on immune function.<ref name=":13">Kraig, E., Linehan, L. A., Liang, H., Romo, T. Q., Liu, Q., Wu, Y., ... & Kellogg Jr, D. L. (2018). A randomized control trial to establish the feasibility and safety of rapamycin treatment in an older human cohort: Immunological, physical performance, and cognitive effects. ''Experimental gerontology'', ''105'', 53-69.</ref> However, laboratory results from this trial suggested that the subjects experienced negative metabolic effects, including a small increase in glycated hemoglobin (within-group p=0.03) and a 40% rise in triglyceride levels (within-group p=0.05).<ref>Lamming, D. Rapamycin and Rapalogs. Preprints 2021, 2021020491 (doi: [https://www.preprints.org/manuscript/202102.0491/v1 10.20944/preprints202102.0491.v1]).</ref> This was a small study with a low dose of rapamycin, dosed over a short duration of 8 weeks. Considering the fact that, based on preclinical animal data, any potential benefit of rapamycin for aging will require long-term dosing, further testing in clinical trials is necessary to better characterize safety.<ref name=":13" />

Clinical trials such as the PEARL study are needed to provide evidence for the safety profile of rapamycin in otherwise healthy older adults.

== Rapalogs ==
Rapalogs are molecules with similar mechanism to rapamycin, primarily via mTORC1 inhibition. These drugs are generally predicted to function similarly to rapamycin in enhancing lifespan and reducing age-related decline in physiological function. However, only one rapalog, everolimus, has published clinical data in this context.<ref>[https://www.thelancet.com/servlet/linkout?suffix=e_1_5_1_2_55_2&dbid=8&doi=10.1016/S2666-7568(20)30068-4&key=24379984&cf= Kaeberlein, M. (2013). mTOR inhibition: from aging to autism and beyond. ''Scientifica'', ''2013''.]</ref> RTB101 has also been described as a selective mTOR inhibitor, but some controversy exists.<ref>Kaeberlein, M. (2020). RTB101 and immune function in the elderly: interpreting an unsuccessful clinical trial. ''Translational Medicine of Aging'', ''4'', 32-34.</ref><ref>Bischof, E., Siow, R. C., Zhavoronkov, A., & Kaeberlein, M. (2021). The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19. ''The Lancet Healthy longevity'', ''2''(2), e105-e111.</ref>

=== mTOR inhibition improves immune function in the elderly ===

In a phase 2 randomized clinical trial published in ''Science Translational Medicine'' in 2014, low-dose TORC1 inhibition with the rapalog everolimus showed improvement in immune function in the elderly. The clinical trial enrolled 218 adults aged ≥65 years, observing decreased incidence of all infections, as well as improved influenza vaccination responses and upregulation of antiviral immunity.<ref name=":17">Mannick, J. B., Del Giudice, G., Lattanzi, M., Valiante, N. M., Praestgaard, J., Huang, B., ... & Klickstein, L. B. (2014). mTOR inhibition improves immune function in the elderly. ''Science translational medicine'', ''6''(268), 268ra179-268ra179.</ref>

Everolimus enhanced the influenza vaccine response by approximately 20% at relatively well tolerated doses.<ref name=":17" /> One mechanism was related to a reduction in the percentage of CD4 and CD8 T cells expressing the programmed death-1 receptor, which has increased expression with age and a major role in inhibiting T cell signaling.<ref name=":17" /> These findings suggest that, at an appopriate dose, mTOR inhibition may improve the age-related decline in immune function in the elderly.

=== TORC1 inhibition enhances immune function and reduces infections in the elderly ===

A phase 2a trial clinical trial randomized 264 older adults to treatment with everolimus and placebo, and was published in ''Science Translational Medicine'' in 2018. The trial showed potential for reducing the effects of immune aging, with improvement in influenza vaccination response in the elderly.<ref name=":1">Mannick, J. B., Morris, M., Hockey, H. U. P., Roma, G., Beibel, M., Kulmatycki, K., ... & Klickstein, L. B. (2018). TORC1 inhibition enhances immune function and reduces infections in the elderly. ''Science translational medicine'', ''10''(449).</ref>[[File:Rtb101 Ph2 Ph3.jpg|thumb|461x461px|A) Number of patients with laboratory-confirmed RTIs of ''any severity'' caused by specific viruses, comparing RTB101 group (blue) versus placebo (grey) in the phase 2b, phase 3, and combined phase 2b/3 RCTs B) Number of patients with laboratory-confirmed RTIs with ''severe symptoms'' caused by specific viruses in the RTB101 group (blue) versus placebo (grey) in the phase 2b, phase 3, and combined phase 2b/3 trials. RTI = respiratory tract infection]]

=== Improving immune function in older adults for respiratory tract infections, including coronaviruses ===
Low-dose mTOR inhibition with dactolisib in a Phase 2b and phase 3 trial in the elderly showed reduced coronavirus (non [[COVID-19]]) incidence, as well as reductions in severe symptoms.<ref name=":12">Mannick, J. B., Teo, G., Bernardo, P., Quinn, D., Russell, K., Klickstein, L., ... & Shergill, S. (2021). Targeting the biology of ageing with mTOR inhibitors to improve immune function in older adults: phase 2b and phase 3 randomised trials. ''The Lancet Healthy Longevity'', ''2''(5), e250-e262.</ref> However, the data remains inconclusive as the study was powered statistically for a reduction in clinically symptomatic respiratory tract infections (RTIs), and not laboratory-confirmed RTIs.<ref name=":12" />

Following the success of two phase 2 clinical trials investigating mTOR inhibition for targeting the aging immune system, dactolisib is currently being pursued for the treatment of COVID-19 in a phase 2a placebo-controlled trial (ClinicalTrials.gov Identifier: NCT04584710, NCT04409327), exploring the potential for preventing severe disease in elderly adults with no symptoms, who have been exposed to [[COVID-19]].<ref name=":12" />

Unpublished data from the phase 2 trial of RTB101 for COVID-19 among nursing home patients treated within 3 days from testing positive saw promising results. None of those treated with RTB101 developed symptoms (n=18), while the placebo treated control group had 4 severe cases of disease and 2 deaths. While this was a statistically significant finding, larger trials are warranted for further evidence of potential benefit.

This trial is being run by the biopharmaceutical company resTORbio and has obtained funding from the National Institute on Aging (NIA/NIH). The studies with dactolisib for COVID-19 is one of several clinical trials in the aging biology field aiming to target aging to improve the aging immune system.<ref name=":12" />

== References ==
<references />
[[Category:Pillar Articles]]
[[Category:Drugs]]

Calorie restriction

2022-08-14T15:36:20Z

Rapacurious: /* CR and Intermittent Fasting */

The physiological decline of an organism, known as aging, is a process highly conserved across the evolutionary tree<ref>Jones, O., Scheuerlein, A., Salguero-Gómez, R., Camarda, C., Schaible, R., & Casper, B. et al. (2013). Diversity of ageing across the tree of life. ''Nature'', ''505''(7482), 169-173. doi: 10.1038/nature12789</ref>. External stressors such as excessive food intake, poor fitness or certain diseases can accelerate biological aging. Reducing calorie intake significantly below the levels of ''ad libitum'' (feeding without restriction) without malnutrition is commonly referred to as calorie restriction (CR) or dietary restriction (DR).<ref name=":19">Bales, C. W., & Kraus, W. E. (2013). Caloric restriction: implications for human cardiometabolic health. ''Journal of cardiopulmonary rehabilitation and prevention'', ''33''(4), 201.</ref>

A number of studies have indicated that CR can increase the lifespan (50-300%) and reduce the onset of age-related diseases in a variety of organisms (e.g. rats, mice, flies, worms, and yeast).<ref name=":0">Flanagan, E. W., Most, J., Mey, J. T., & Redman, L. M. (2020). Calorie Restriction and Aging in Humans. ''Annual Review of Nutrition'', ''40'', 105-133.</ref> There is some evidence from human epidemiological and clinical trial data suggesting that CR could increase healthy lifespan by 1 to 5 years.<ref name=":0" />

Care should be taken when using CR as a means to increase lifespan and prevent age-related diseases. It is important to recognize that scientists point to the benefits of CR only when avoiding malnutrition and when performed under adequate nutrition.<ref name=":20">[https://doi.org/10.1016/j.arr.2010.05.002 Cerqueira, F., & Kowaltowski, A. (2010). Commonly adopted caloric restriction protocols often involve malnutrition. ''Ageing Research Reviews'', ''9''(4), 424-430. doi: 10.1016/j.arr.2010.05.002]</ref> Nutrient deficiencies are associated with various health deficits, and consuming less calories than recommended can also be detrimental. There is also concern that reductions in body fat mass could affect muscle bone and tissue functionality.<ref name=":0" /> Thus, it is important to have sufficient quality macronutrient intake along with CR.

Additionally, there are risks associated to impaired immune function during CR interventions, an example of a potential trade-off.<ref name=":0" /> There may be utility in combining CR with other interventions to maximize healthy longevity, but more data is needed from both animal and human studies.

Several mice studies have shown that different genetic backgrounds may substantially influence the response to CR.<ref name=":5" /> This means that while some mice strains obtain lifespan benefits, others may attain no benefit or even experience harmful consequences.

== Evidence ==
CR is the most widely researched intervention for slowing aging and preventing age-related diseases. Clive McCay first published his groundbreaking research in 1935 with the observation that rats with restricted diets experienced a 33% increase in lifespan.<ref>McCay, C. M., Crowell, M. F., & Maynard, L. A. (1935). The effect of retarded growth upon the length of life span and upon the ultimate body size: one figure. ''The journal of Nutrition'', ''10''(1), 63-79.</ref>

Similar survival experiments have shown that DR can increase the median and maximum lifespan of a variety of other organisms. Below we discuss in more details findings in each species:

=== Worms ===
''Caenorhabditis elegans'' is a roundworm nematode widely used as an aging animal model.<ref>Lakowski, B., & Hekimi, S. (1998). The genetics of caloric restriction in Caenorhabditis elegans. ''Proceedings of the National Academy of Sciences'', ''95''(22), [tel:13091-13096 13091-13096].</ref> Mutations in "''eat''" genes disrupt the function of the pharynx and the feeding behaviour of the worm, leading to partial starvation. ''Eat'' mutations are therefore considered CR-mimetics and can lengthen the lifespan of worms by up to 50%.<ref>Lakowski, B., & Hekimi, S. (1998). The genetics of caloric restriction in <nowiki><i>Caenorhabditis elegans</i></nowiki>. ''Proceedings Of The National Academy Of Sciences'', ''95''(22), [tel:13091-13096 13091-13096]. doi: 10.1073/pnas.95.22.13091</ref> The most studied "''eat''" gene in C. elegans, ''eat-2,'' extends lifespan through a mechanism independent of the insulin-signalling pathway, as it does not require the transcription factor [[FOXO longevity genes|''daf-16/FOXO'']] (a central component of the insulin signalling pathway) to extend lifespan. ''Eat-2'' mutants, as well as wild-type worms under CR, require the transcription factor ''pha-4/FOXA'' for the associated lifespan extension phenotype. More specifically, ''pha-4/FOXA'' is required in the intestinal tissue, but not in other tissues such as the nervous tissue, muscle or hypodermis.<ref>Panowski, S., Wolff, S., Aguilaniu, H. ''et al.'' (2007). PHA-4/Foxa mediates diet-restriction-induced longevity of ''C. elegans''. ''Nature'' 447, 550–555. <nowiki>https://doi.org/10.1038/nature05837</nowiki></ref>

In another study, it was found that when C. ''elegans'' experiences dietary restriction early during development, proteostasis is enhanced and adult lifespan is increased.<ref>Matai, L., Sarkar, G., Chamoli, M., Malik, Y., Kumar, S., & Rautela, U. et al. (2019). Dietary restriction improves proteostasis and increases life span through endoplasmic reticulum hormesis. ''Proceedings Of The National Academy Of Sciences'', ''116''(35), [tel:17383-17392 17383-17392]. doi: 10.1073/pnas.1900055116</ref> Similarly, both dietary restriction and dietary deprivation complete removal of food) in adulthood is reported to increase lifespan and to enhance thermotolerance and resistance to oxidative stress.<ref>Lee, G., Wilson, M., Zhu, M., Wolkow, C., de Cabo, R., Ingram, D., & Zou, S. (2006). Dietary deprivation extends lifespan in Caenorhabditis elegans. ''Aging Cell'', ''5''(6), 515-524. doi: 10.1111/j.1474-9726.2006.00241.x</ref>

=== Mice ===
A caloric-restriction experiment was conducted on wild mice to see if they would experience similar results as genetically bred lab mice.<ref>Harper, J., Leathers, C., & Austad, S. (2006). Does caloric restriction extend life in wild mice?. ''Aging Cell'', ''5''(6), 441-449. doi: 10.1111/j.1474-9726.2006.00236.x</ref> Whilst the 8.1% of longest-lived wild mice belonged to the CR test group, there was no robust longevity difference in mean lifespan between the groups. However, there was an anticancer effect in the CR group, as seen in other experiments with laboratory bred mice. Authors argued that strong differences in longevity were not noted possibly because wild animals have a higher genetic variation than inbred mice, which could affect CR strength.

In another study, it was noted that caloric restriction increased working memory in mice.<ref>Kuhla, A., Lange, S., Holzmann, C., Maass, F., Petersen, J., Vollmar, B., & Wree, A. (2013). Lifelong Caloric Restriction Increases Working Memory in Mice. ''Plos ONE'', ''8''(7), e68778. doi: 10.1371/journal.pone.0068778</ref> Male mice that experienced long periods of fasting between meals were found to live longer and healthier lifespans, regardless of the types of food they ate.

Inbred mice have shown to benefit significantly less from CR interventions than non-inbred mice, with some inbred mice strains not benefiting at all from CR.<ref name=":16">Swindell, W. (2012). Dietary restriction in rats and mice: A meta-analysis and review of the evidence for genotype-dependent effects on lifespan. ''Ageing Research Reviews'', ''11''(2), 254-270. doi: 10.1016/j.arr.2011.12.006</ref> Therefore, this suggests rodent studies might be potentially biased when conducting experiments in laboratory inbred mice and encourages the diversification of CR studies in a wider genetic background.

=== Dogs ===

==== Purina Lifespan Study ====
The Purina Lifespan Study was performed on Labrador retrievers randomly assigned to either 25% caloric restriction or to control feeding, offering the same diet and only differing by quantity.<ref name=":6">Lawler, D., Larson, B., Ballam, J., Smith, G., Biery, D., & Evans, R. et al. (2007). Diet restriction and ageing in the dog: major observations over two decades. ''British Journal Of Nutrition'', ''99''(4), 793-805. doi: 10.1017/s0007114507871686</ref><ref name=":8">Kealy, R. D., Lawler, D. F., Ballam, J. M., Mantz, S. L., Biery, D. N., Greeley, E. H., ... & Stowe, H. D. (2002). Effects of diet restriction on life span and age-related changes in dogs. ''Journal of the American Veterinary Medical Association'', ''220''(9), 1315-1320.</ref> Over 14 years of follow up, there was a 1.8 year extension in median lifespan along with several improved health markers, such as delayed osteoarthritis.<ref name=":6" /><ref name=":8" /> Whilst various measures of immune function are expected to decline with age,<ref>Greeley, E., Ballam, J., Harrison, J., Kealy, R., Lawler, D., & Segre, M. (2001). The influence of age and gender on the immune system: a longitudinal study in Labrador Retriever dogs. ''Veterinary Immunology And Immunopathology'', ''82''(1-2), 57-71. doi: 10.1016/s0165-2427(01)00336-1</ref> the study showed that total lymphocytes, T-cells and CD8 cells did not decline in the CR group, in contrast to observed declines in the immune system function of the control diet group.<ref name=":6" />

==== Dog Aging Project ====
The [[Rapamycin|Dog Aging Project]] is an initiative that is studying thousands of dogs over their lifetimes to understand which environmental and genetic factors influence healthy aging.<ref name=":7" /> In an early study of 10.474 companion dogs, one-time daily feeding versus more frequent feeding was associated with better measures across multiple domains of health.<ref name=":7" /> This included lower scores on the Canine Cognitive Dysfunction Rating Scale, and lower odds of having gastrointestinal, dental, orthopedic, kidney, urinary, liver and pancreas disorders. The authors suggest that while this preliminary data is not sufficient to support recommendations for meal timing in pet dogs, this data might help guide future research into dietary variables that affect health.<ref name=":7">Bray, E. E., Zheng, Z., Tolbert, M. K., McCoy, B. M., Kaeberlein, M., & Kerr, K. F. (2022). Once-daily feeding is associated with better health in companion dogs: results from the Dog Aging Project. ''GeroScience'', 1-12.</ref>

=== Primates ===

==== Restrikal study (2006) ====
The Restrikal study, initiated in 2006, studied the effect of chronic 30% CR in the grey mouse lemur primate, ''Microcebus murinus''.<ref name=":4">Pifferi, F., Terrien, J., Marchal, J., Dal-Pan, A., Djelti, F., Hardy, I., ... & Aujard, F. (2018). Caloric restriction increases lifespan but affects brain integrity in grey mouse lemur primates. ''Communications biology'', ''1''(1), 1-8.</ref> Results of the study indicated that CR prolonged lifespan by 50%, from 6.4 to 9.6 years, but affected brain structural integrity.<ref name=":4" /> It was observed that gray matter integrity in the cerebrum was compromised by CR, yet importantly, this did not result in any apparent changes to cognitive function.

==== NIA (2012) & Wisconsin NPRC (2014) studies controversy ====
The National Institute on Aging (NIA) study in Maryland, USA, performed CR in rhesus monkeys and saw no differences between survival of monkeys fed control versus calorie-restricted diets.<ref name=":17">Mattison, J. A., Roth, G. S., Beasley, T. M., Tilmont, E. M., Handy, A. M., Herbert, R. L., ... & De Cabo, R. (2012). Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study. ''Nature'', ''489''(7415), 318-321.</ref> The diet of controls in this study was not reported as fully ''ad libitum'', but rather control monkeys were subject to a slight dietary restriction to prevent obesity.

On the other hand, in the Wisconsin National Primate Research Centre (WNPRC) study, rhesus monkeys subjected to long-term 30% dietary restriction showed a significantly reduced risk of all-cause mortality and age-related mortality compared to control group monkeys. This suggested the benefits of CR on aging might be conserved in primates.<ref name=":18">Colman, R. J., Beasley, T. M., Kemnitz, J. W., Johnson, S. C., Weindruch, R., & Anderson, R. M. (2014). Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys. ''Nature communications'', ''5''(1), 1-5.</ref>

Given that the Wisconsin and NIA primate studies found contradictory results, researchers have attempted to determine why slowed aging was only demonstrated in the Wisconsin study. The observed differences between these two studies is particularly controversial because the control primates in the NIA study lived longer than the CR group in the Wisconsin study, suggesting differences in methodology played an important role.

Some have suggested that diet composition is important, due to clear differences in feeding quality and composition between the Wisconsin and NIA studies. A key difference is certainly the fact that the Wisconsin study subjected monkeys to strict ''ad libitum'' in the control group, whilst the NIA study did not in order to prevent obesity. The latter is generally considered a better controlled experiment.

=== Humans ===
There is currently no definite evidence that calorie restriction extends healthy human lifespan.<ref name=":5">Lee, M. B., Hill, C. M., Bitto, A., & Kaeberlein, M. (2021). Antiaging diets: Separating fact from fiction. ''Science'', ''374''(6570), eabe7365.</ref> However, there is early clinical evidence suggesting that CR without malnutrition may lead to various health benefits related to aging, based on several randomized controlled trials. In many human studies, CR is defined as a restriction of calories by ≥10% compared to feeding without restriction (''ad libitum'').<ref name=":19" />

'''The population of Okinawa'''

Studies into certain populations known for their exceptional longevity, such as in Okinawa - a small island of Japan - have provided some insights into potential lifestyle determinants of longevity. Okinawans have long been recognized as one of the longest-lived populations on the planet, and this is typically attributed to their diet (fish and vegetables). However, more recently, some attention in the scientific community has deviated from the contents of Okinawan’s diets and focused, instead, on their caloric deficits. Six generations of Okinawans aged 65+ were studied; their diet composition, energy intake and expenditure, and survival patterns were analyzed, among many other factors. The results lent support to the wide-ranging health benefits of caloric restriction in humans. Some researchers have speculated that the introduction of Westernized diets may in part explain recent decreases in Okinawan population lifespan.<ref name=":0" />

'''Biosphere-II'''

The Biosphere II experiment was an ecological investigation that provided an unexpected opportunity to measure the effects of CR.<ref>Walford, R., Mock, D., Verdery, R., & MacCallum, T. (2002). Calorie Restriction in Biosphere 2: Alterations in Physiologic, Hematologic, Hormonal, and Biochemical Parameters in Humans Restricted for a 2-Year Period. ''The Journals Of Gerontology Series A: Biological Sciences And Medical Sciences'', ''57''(6), B211-B224. doi: 10.1093/gerona/57.6.b211</ref> Eight volunteers were kept in an ecological ecosystem for two years and allowed to harvest 85% of their food. The food consisted mainly of fruits, vegetables, grains and minimal protein. During the experiment, because of food scarcity, the energy intake of the volunteers decreased by 38% for 6 months. After leaving the experiment the volunteers had a 6% slowing of metabolism which lasted for another 6 months.

Years later, a Biosphere-II participant founded the ''CR Society International,'' which consists of a group of volunteers that have chosen to restrict their calorie intake around 30% for a period of 3 to 15 years.<ref>Fontana, L., Meyer, T., Klein, S., & Holloszy, J. (2004). Long-term calorie restriction is highly effective in reducing the risk for atherosclerosis in humans. ''Proceedings Of The National Academy Of Sciences'', ''101''(17), [tel:6659-6663 6659-6663]. doi: 10.1073/pnas.0308291101</ref> Individuals of the CR society appear leaner, have lower body fat, better cardiometabolic health and lower inflammation. However, this data is sparse and largely limited to self-reports.

'''CALERIE trials'''

The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) research network has produced one of the most rigorous clinical studies conducted in humans. Over a period of nine years, three pilot trials were conducted followed by a randomized study (CALERIE 2).<ref name=":1">Kraus, W. E., Bhapkar, M., Huffman, K. M., Pieper, C. F., Das, S. K., Redman, L. M., ... & CALERIE Investigators. (2019). 2 years of calorie restriction and cardiometabolic risk (CALERIE): exploratory outcomes of a multicentre, phase 2, randomised controlled trial. ''The lancet Diabetes & endocrinology'', ''7''(9), 673-683.</ref><ref name=":2">Rickman, A. D., Williamson, D. A., Martin, C. K., Gilhooly, C. H., Stein, R. I., Bales, C. W., ... & Das, S. K. (2011). The CALERIE Study: design and methods of an innovative 25% caloric restriction intervention. ''Contemporary clinical trials'', ''32''(6), 874-881.</ref>

During phase 1 of the trial, three differing degrees of CR (20%, 25%, and 30%) were tested in a variety of age groups with an overweight BMI status. The trial lasted for 6 – 12 months, and the studies were used to develop and advance the following Phase 2 trial.

In Phase 2 of CALERIE, participants were able to restrict caloric intake by 11.9% and experienced ~10% weight loss over two years, despite the identified target of 25% CR. It must be noted that the level of CR achieved in this study required intensive intervention, involving personalized treatments, algorithmic/computer tracking, and various educational initiatives.<ref name=":2" /> Therefore, the feasibility of such a CR intervention in the real world is something that remains uncharacterized. However, despite participants in the CR group achieving a lower CR target than intended, various improvements to health were noted. The trial resulted in lower levels of T3 and TNF-ɑ, while also reducing certain cardiometabolic risk factors.<ref name=":1" />

Additional analyses suggested a slow down in the rate of biological aging and found that weight loss did not appear to account for these effects.<ref name=":3">Belsky, D. W., Huffman, K. M., Pieper, C. F., Shalev, I., & Kraus, W. E. (2018). Change in the rate of biological aging in response to caloric restriction: CALERIE Biobank analysis. ''The Journals of Gerontology: Series A'', ''73''(1), 4-10.</ref> The authors highlighted that, based on prior knowledge that a divergence in biological aging trajectories can be observed as early as early adulthood, CR may be more effective in humans when started young.<ref>Belsky, D. W., Caspi, A., Houts, R., Cohen, H. J., Corcoran, D. L., Danese, A., ... & Moffitt, T. E. (2015). Quantification of biological aging in young adults. ''Proceedings of the National Academy of Sciences'', ''112''(30), E4104-E4110.</ref> Moreover, potential CR-related toxicities were posited to be better tolerated in younger adults.<ref name=":3" />

'''CR and immune function - randomized controlled trial'''

One clinical study investigated moderate CR versus ''ad-libitum'' feeding over 2 years. It was found that CR without malnutrition may induce health benefits without impairing cell-mediated immunity or increasing infection risk in non-obese humans.<ref>Meydani, S. N., Das, S. K., Pieper, C. F., Lewis, M. R., Klein, S., Dixit, V. D., ... & Fontana, L. (2016). Long-term moderate calorie restriction inhibits inflammation without impairing cell-mediated immunity: a randomized controlled trial in non-obese humans. ''Aging (Albany NY)'', ''8''(7), 1416.</ref>

== Underlying biological mechanisms ==
Consuming extra calories can lead to cellular glycotoxicity and lipotoxicity, which causes inflammation and oxidative stress and thus increases the risk of age-related diseases (e.g. cancer, diabetes, cardiovascular disorders).<ref name=":0" /> Some proposed health benefits of CR include preservation of cognition, protection of colon health and reduced risk of arthritis, amongst others.

Evidence suggests that CR may lead to a variety of health benefits via the following biological pathways:<ref name=":0" />

* Inhibition of mTOR pathway and consequent induction of a[[Autophagy|utophagy]], a specific process that recycles cellular waste.
*Activation of known pro-longevity pathways such as FOXO/AMPK/SIRT, which are evolutionarily conserved across various species.
*Increase in coenzyme Q10 (CoQ) dependent reductases within the plasma membrane, thus protecting phospholipids and preventing the lipid peroxidation reaction progression.
*Reduction in oxidative damage due to a decreased production of Reactive Oxygen Species (ROS).
*Decrease in the systemic risk factors for cardiovascular disease (glucose levels, blood pressure, plasma lipid levels).
* Alteration in the sympathetic nervous system, as well as the neuroendocrine system in lab animals and, sometimes, humans.

== Caveats of caloric restriction ==
An exhaustive review of calorie restriction experiments in rodents highlighted some of the most common questions and caveats of CR:<ref name=":11">Ingram, D., & de Cabo, R. (2017). Calorie restriction in rodents: Caveats to consider. ''Ageing Research Reviews'', ''39'', 15-28. doi: 10.1016/j.arr.2017.05.008</ref>

===== 1. Until what age is CR effective? =====
One important aspect to consider is whether CR interventions are effective regardless of age of the individual. Whilst the question remains open as of today, available evidence suggests that performing CR in advanced age leads to a wide range of health benefits (especially in motor function) and is able to extend lifespan. However, it seems that late-life CR can extend lifespan to a significantly lesser degree than early-adulthood CR, although definitive evidence is missing.<ref name=":11" /><ref>[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC397416/ Dhahbi, J. M., Kim, H. J., Mote, P. L., Beaver, R. J., & Spindler, S. R. (2004). Temporal linkage between the phenotypic and genomic responses to caloric restriction. ''Proceedings of the National Academy of Sciences'', ''101''(15), 5524-5529.]</ref>

In humans, there is concern that late-life CR may exacerbate age-related muscle loss (sarcopenia) and potentiate the effects of falls.<ref name=":9">Flanagan, E. W., Most, J., Mey, J. T., & Redman, L. M. (2020). Calorie restriction and aging in humans. ''Annual review of nutrition'', ''40'', 105.</ref> While various pre-clinical studies have shown potential for treating or preventing sarcopenia, there is a lack of clinical evidence supporting its use in older, otherwise non-obese patients.<ref name=":9" /><ref>Liu, P., Li, Y., & Ma, L. (2021). Caloric Restriction May Help Delay the Onset of Frailty and Support Frailty Management. ''Frontiers in Nutrition'', ''8''.</ref><ref>Racette, S. B., Weiss, E. P., Villareal, D. T., Arif, H., Steger-May, K., Schechtman, K. B., ... & Holloszy, J. O. (2006). One year of caloric restriction in humans: feasibility and effects on body composition and abdominal adipose tissue. ''The Journals of Gerontology Series A: Biological Sciences and Medical Sciences'', ''61''(9), 943-950.

Chicago</ref>

===== 2. Does CR improve cognitive decline? =====
CR has been shown to attenuate cognitive decline in mice models of Alzheimer's disease and to dramatically improve the behavioral phenotype of progeroid DNA-repair deficient mice.<ref>Dhurandhar, E., Allison, D., van Groen, T., & Kadish, I. (2013). Hunger in the Absence of Caloric Restriction Improves Cognition and Attenuates Alzheimer's Disease Pathology in a Mouse Model. ''Plos ONE'', ''8''(4), e60437. doi: 10.1371/journal.pone.0060437</ref><ref>Vermeij, W., Dollé, M., Reiling, E., Jaarsma, D., Payan-Gomez, C., & Bombardieri, C. et al. (2016). Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice. ''Nature'', ''537''(7620), 427-431. doi: 10.1038/nature19329</ref>

Despite this encouraging evidence, whether CR improves cognitive function in wild-type backgrounds appears largely inconsistent across experiments. A main issue might be the lack of systematic research and the range of differences in CR protocols, which has so far hindered extracting definitive conclusions. Similarly, several studies have shown inconclusive results in other species, such as in the fly ''Drosophila'' ''Melanogaster'' and the grey mouse lemur primate.<ref>Kerr, F., Augustin, H., Piper, M. D., Gandy, C., Allen, M. J., Lovestone, S., & Partridge, L. (2011). Dietary restriction delays aging, but not neuronal dysfunction, in Drosophila models of Alzheimer's disease. ''Neurobiology of aging'', ''32''(11), 1977-1989.</ref><ref>Pifferi, F., Terrien, J., Marchal, J., Dal-Pan, A., Djelti, F., Hardy, I., ... & Aujard, F. (2018). Caloric restriction increases lifespan but affects brain integrity in grey mouse lemur primates. ''Communications biology'', ''1''(1), 1-8.</ref>

In humans, the CALERIE trial included multiple tests of cognitive function and found that in non-obese, healthy adults, working memory was slightly improved after 2 years of CR.<ref name=":15">Leclerc, E., Trevizol, A. P., Grigolon, R. B., Subramaniapillai, M., McIntyre, R. S., Brietzke, E., & Mansur, R. B. (2020). The effect of caloric restriction on working memory in healthy non-obese adults. ''CNS spectrums'', ''25''(1), 2-8.</ref> However, this was a post-hoc analysis, which is not sufficient evidence to support causation.<ref name=":15" />

===== 3. What are the deleterious effects of CR? =====

====== Bone health ======
In rodents, conventional 30% CR interventions have been associated to reduced body mass and reduced bone density, specially when CR is initiated in early life but also when initiated at mature or older ages.<ref>Baek, K., Barlow, A., Allen, M., & Bloomfield, S. (2008). Food restriction and simulated microgravity: effects on bone and serum leptin. ''Journal Of Applied Physiology'', ''104''(4), 1086-1093. doi: 10.1152/japplphysiol.01209.2007</ref><ref>Banu, J., Orhii, P., Okafor, M., Wang, L., & Kalu, D. (2001). Analysis of the effects of growth hormone, exercise and food restriction on cancellous bone in different bone sites in middle-aged female rats. ''Mechanisms Of Ageing And Development'', ''122''(8), 849-864. doi: 10.1016/s0047-6374(01)00243-3</ref> More sophisticated studies have indicated that CR does not negatively impact bone material properties, despite its association to reduced bone size and decreased whole-bone strength.<ref>Huang, T., & Ables, G. (2016). Dietary restrictions, bone density, and bone quality. ''Annals Of The New York Academy Of Sciences'', ''1363''(1), 26-39. doi: 10.1111/nyas.13004</ref>

====== Wound healing ======
Experiments in both mice and rats has so far shown that animals under CR have a diminished capacity for skin wound healing than normally fed controls.<ref name=":12">Hunt, N., Li, G., Zhu, M., Levette, A., Chachich, M., & Spangler, E. et al. (2011). Effect of calorie restriction and refeeding on skin wound healing in the rat. ''AGE'', ''34''(6), 1453-1458. doi: 10.1007/s11357-011-9321-6</ref><ref name=":13">Reed, M., Penn, P., Li, Y., Birnbaum, R., Vernon, R., & Johnson, T. et al. (1996). Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice. ''Mechanisms Of Ageing And Development'', ''89''(1), 21-43. doi: 10.1016/0047-6374(96)01737-x</ref> This appears to be consistent ''in vitro'', where CR mice-derived cells were reported to have reduced proliferation compared to control cells.<ref>Hsieh, E., Chai, C., & Hellerstein, M. (2005). Effects of caloric restriction on cell proliferation in several tissues in mice: role of intermittent feeding. ''American Journal Of Physiology-Endocrinology And Metabolism'', ''288''(5), E965-E972. doi: 10.1152/ajpendo.00368.2004</ref> Importantly, ''in vivo'' experiments found that wound healing capacity was restored to that of control levels a short period after rodents were normally fed again.<ref name=":12" /><ref name=":13" />

====== Immune response ======
Several studies have shown that mice undergoing CR have an increased risk of infection than those on ''ad libitum'' diets. This is presumably due to a less efficient immune response, given that CR animals are not able to respond as well to the higher metabolic demands that an infection supposes.<ref>Kristan, D. (2007). Chronic calorie restriction increases susceptibility of laboratory mice (Mus musculus) to a primary intestinal parasite infection. ''Aging Cell'', ''6''(6), 817-825. doi: 10.1111/j.1474-9726.2007.00345.x</ref><ref>Kristan, D. (2008). Calorie restriction and susceptibility to intact pathogens. ''AGE'', ''30''(2-3), 147-156. doi: 10.1007/s11357-008-9056-1</ref> However, similar to wound healing experiments, animals showed a recovered capacity to fight infection shortly after being re-fed. On the contrary, other studies have reported no deleterious effects of CR in response to infection or even beneficial ones.<ref>Hasegawa, A., Iwasaka, H., Hagiwara, S., Asai, N., Nishida, T., & Noguchi, T. (2012). Alternate Day Calorie Restriction Improves Systemic Inflammation in a Mouse Model of Sepsis Induced by Cecal Ligation and Puncture. ''Journal Of Surgical Research'', ''174''(1), 136-141. doi: 10.1016/j.jss.2010.11.883</ref>

===== 4. Are feeding times in CR important? =====

====== Time of feeding ======
The benefits of caloric restriction in mice appear to be affected by the timing of feeding during the day. As nocturnal animals, mice that underwent CR during their normally active feeding period (night time) showed increased health benefits compared to mice undergoing CR during their rest time (daylight), as measured by structural changes in the gut microbiota.<ref>Zhang, L., Xue, X., Zhai, R., Yang, X., Li, H., Zhao, L., & Zhang, C. (2019). Timing of Calorie Restriction in Mice Impacts Host Metabolic Phenotype with Correlative Changes in Gut Microbiota. ''Msystems'', ''4''(6). doi: 10.1128/msystems.00348-19</ref> This showcases the important link between circadian clocks, CR interventions and potentially lifespan.

====== CR and Intermittent Fasting ======
''See the full article on [[fasting]].''

Intermittent fasting (IF) has attained popular interest in recent years for its various potential health benefits, including for treating disease and extending lifespan.<ref>Lee, M. B., Hill, C. M., Bitto, A., & Kaeberlein, M. (2021). Antiaging diets: Separating fact from fiction. ''Science'', ''374''(6570), eabe7365.

Chicago</ref><ref name=":10">Longo, V. D., Di Tano, M., Mattson, M. P., & Guidi, N. (2021). Intermittent and periodic fasting, longevity and disease. ''Nature aging'', ''1''(1), 47-59.</ref> Some preclinical evidence shows that certain IF regimens can prevent the onset of many age-related diseases. However, IF is not always associated with benefits in healthspan and may increase or decrease lifespan.<ref name=":10" /> It has been proposed that at least part of the lifespan extending effect of CR in humans is related to fasting, and in rodents fasting is required for a CR diet to protect from frailty and extend lifespan.<ref name=":10" /><ref>Pak, H. H., Haws, S. A., Green, C. L., Koller, M., Lavarias, M. T., Richardson, N. E., ... & Lamming, D. W. (2021). Fasting drives the metabolic, molecular and geroprotective effects of a calorie-restricted diet in mice. ''Nature metabolism'', ''3''(10), 1327-1341.</ref> In rodent models, the evidence for IF preventing cancer development or growth is ambiguous, with studies showing no effect or potential harm with IF. More studies are required to better understand IF, both for preclinical and clinical research.<ref>Clifton, K. K., Ma, C. X., Fontana, L., & Peterson, L. L. (2021). Intermittent fasting in the prevention and treatment of cancer. ''CA: a cancer journal for clinicians'', ''71''(6), 527-546.</ref>

A recent study in China randomized 139 obese adults to either calorie restriction alone or to calorie restriction with time-restricted eating (a 16-hour intermittent fast and a 8-hour period for eating).<ref>Liu, D., Huang, Y., Huang, C., Yang, S., Wei, X., & Zhang, P. et al. (2022). Calorie Restriction with or without Time-Restricted Eating in Weight Loss. ''New England Journal Of Medicine'', ''386''(16), 1495-1504. doi: 10.1056/nejmoa2114833</ref> After one year, both groups had lost 7-10% of body weight and showed healthier markers for blood sugar, blood fat levels and insulin sensitivity. Importantly, there was no statistically significant difference between both groups, suggesting calorie restriction is responsible for the health-associated benefits and that intermittent fasting has no added benefits to CR diets.

===== 5. How long should CR interventions last? =====
Current evidence in rodents suggests that short-term CR (considered as interventions ranging from 1 day to a few months) can still have beneficial effects in health, similar to traditional long-term CR.<ref>Robertson, L., & Mitchell, J. (2013). Benefits of short-term dietary restriction in mammals. ''Experimental Gerontology'', ''48''(10), 1043-1048. doi: 10.1016/j.exger.2013.01.009</ref> Short-term CR has also been associated to increased health biomarkers, to improve diseased-states in models of hypertensive rats and to enhance the benefits of chemotherapy in cancer mouse models.<ref>Chiba, T., & Ezaki, O. (2010). Dietary restriction suppresses inflammation and delays the onset of stroke in stroke-prone spontaneously hypertensive rats. ''Biochemical And Biophysical Research Communications'', ''399''(1), 98-103. doi: 10.1016/j.bbrc.2010.07.048</ref><ref>Mitchell, J., Verweij, M., Brand, K., van de Ven, M., Goemaere, N., & van den Engel, S. et al. (2010). Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice. ''Aging Cell'', ''9''(1), 40-53. doi: 10.1111/j.1474-9726.2009.00532.x</ref>

===== 6. What are the genotype and gender effects on CR? =====
There is strong evidence that genotype and gender within the same species can have a dramatic effect on the efficiency of CR. A meta-analysis from mice studies performed between 1934 and 2012 revealed that lifespan extension could vary within gender of the same strain and also between different strains, with the degree of lifespan extension ranging between 4 to 27%.<ref>Swindell, W. (2012). Dietary restriction in rats and mice: A meta-analysis and review of the evidence for genotype-dependent effects on lifespan. ''Ageing Research Reviews'', ''11''(2), 254-270. doi: 10.1016/j.arr.2011.12.006</ref><ref>Mitchell, S., Madrigal-Matute, J., Scheibye-Knudsen, M., Fang, E., Aon, M., & González-Reyes, J. et al. (2016). Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice. ''Cell Metabolism'', ''23''(6), 1093-1112. doi: 10.1016/j.cmet.2016.05.027</ref> Strains belonging to the lower end of percentage of lifespan extension are often recombinant inbred strains.<ref name=":16" /> Other studies have demonstrated that certain mice genotypes are highly unresponsive to CR interventions, although it is argued that different genotypes might be responsive to different strengths of CR.<ref>Liao, C., Rikke, B., Johnson, T., Diaz, V., & Nelson, J. (2010). Genetic variation in the murine lifespan response to dietary restriction: from life extension to life shortening. ''Aging Cell'', ''9''(1), 92-95. doi: 10.1111/j.1474-9726.2009.00533.x</ref><ref>Rikke, B., Liao, C., McQueen, M., Nelson, J., & Johnson, T. (2010). Genetic dissection of dietary restriction in mice supports the metabolic efficiency model of life extension. ''Experimental Gerontology'', ''45''(9), 691-701. doi: 10.1016/j.exger.2010.04.008</ref> As previously discussed in [[Calorie restriction#Primates|primates]], similar observations have been reported in rhesus monkeys undergoing 30% CR, where independent groups reported similar health benefits but largely dissimilar survival curves, highlighthing the complexity of genotypic and/or environmental factors in the efficiency of CR.<ref name=":17" /><ref name=":18" />

===== 7. Is diet composition important in CR? =====
''See the full article on [[protein restriction]].''

Diet composition in CR is likely to be important. As mentioned previously, there is a general agreement that CR should reduce the total amount of calories ingested whilst avoiding malnutrition (ie. without reducing protein or macronutrient intake), in order to maximise health benefits.<ref name=":19" /><ref name=":20" /> In fact, an exhaustive review of literature suggested that most commonly adopted CR protocols led to malnutrition in rodents, which they argue might have added to the observed discrepancies in survival curves across CR interventions.<ref>Cerqueira, F., & Kowaltowski, A. (2010). Commonly adopted caloric restriction protocols often involve malnutrition. ''Ageing Research Reviews'', ''9''(4), 424-430. doi: 10.1016/j.arr.2010.05.002</ref>

Some scientists have proposed that [[protein restriction]] (PR) in particular is responsible for the benefits observed in CR.<ref>Simpson, S., & Raubenheimer, D. (2007). Caloric Restriction and Aging Revisited: The Need for a Geometric Analysis of the Nutritional Bases of Aging. ''The Journals Of Gerontology Series A: Biological Sciences And Medical Sciences'', ''62''(7), 707-713. doi: 10.1093/gerona/62.7.707</ref><ref>Raubenheimer, D., Simpson, S., & Tait, A. (2012). Match and mismatch: conservation physiology, nutritional ecology and the timescales of biological adaptation. ''Philosophical Transactions Of The Royal Society B: Biological Sciences'', ''367''(1596), 1628-1646. doi: 10.1098/rstb.2012.0007</ref> However, more recent studies reviewing all published data since the early 1930s until 2016 concluded that lifespan extension in rodents is due to CR alone and not due to a reduction of protein or any other macronutrients.<ref>Speakman, J., Mitchell, S., & Mazidi, M. (2016). Calories or protein? The effect of dietary restriction on lifespan in rodents is explained by calories alone. ''Experimental Gerontology'', ''86'', 28-38. doi: 10.1016/j.exger.2016.03.011</ref><ref>Pamplona, R., & Barja, G. (2006). Mitochondrial oxidative stress, aging and caloric restriction: The protein and methionine connection. ''Biochimica Et Biophysica Acta (BBA) - Bioenergetics'', ''1757''(5-6), 496-508. doi: 10.1016/j.bbabio.2006.01.009</ref>

== Controversies of calorie restriction research ==
There are several criticisms against CR, some of which are highlighted by Sohal and Forster (2014) in “''Caloric Restriction and the Aging Process: A Critique''”.<ref>Sohal, R. S., & Forster, M. J. (2014). Caloric restriction and the aging process: a critique. ''Free radical biology & medicine'', ''73'', 366–382. <nowiki>https://doi.org/10.1016/j.freeradbiomed.2014.05.015</nowiki></ref> The authors highlight that there is a large disparity in CR-related longevity increases: namely, that longevity effects are not universal and sometimes are not shared across genetic strains of the same species. Moreover, control animals in widely-cited caloric restriction studies were mostly fed ''ad libitum'', causing them to become overweight and vulnerable to disease and early death. Therefore the relative benefit in the CR group was exaggerated compared to control subjects. In other words, animals with CR diets may live relatively longer because the control animals were dying from complications of excess feeding.

Another challenge related to CR as an effective intervention for human aging is the difficulty in compliance over long periods of time.<ref name=":0" /> Concerns over mental and sexual health have also been raised with more severe CR. There are concerns over the loss of weight and fat mass in younger people practicing CR. Exercising along with CR and good nutrition (high protein diet) appears to be highly beneficial for loss of free fat.<ref name=":0" /> New nutritional approaches such as intermittent fasting have emerged. However, there is comparatively limited research on the topic, with CR being the most well-studied nutritional intervention for healthy aging. Furthermore, studies in worms showed that Allantoin, rapamycin, TSA and LY-294002 led to a slower decline in pharyngeal pumping, indicating a reduced aging rate.<ref name=":14">Calvert, S., Tacutu, R., Sharifi, S., Teixeira, R., Ghosh, P., & de Magalhães, J. P. (2016). A network pharmacology approach reveals new candidate caloric restriction mimetics in C. elegans. ''Aging cell'', ''15''(2), 256-266.</ref> Thus, the study uncovered that not only could drug treatments mimicking CR increase longevity, but they could also improve the organism’s healthfulness.<ref name=":14" />

It is important to note that researchers are increasingly aware that CR or strict intermittent fasting are not a “one size fits all”, but rather an efficient strategy for certain individuals in specific metabolic contexts. For instance, some studies have shown that two people's glucose responses are significantly different even after eating the same food.<ref>Zeevi, D., Korem, T., Zmora, N., Israeli, D., Rothschild, D., & Weinberger, A. et al. (2015). Personalized Nutrition by Prediction of Glycemic Responses. ''Cell'', ''163''(5), 1079-1094. doi: 10.1016/j.cell.2015.11.001</ref> Supporting these findings, companies like [https://www.lumen.me/metabolic-flexibility Lumen Metabolism] and [https://www.levelshealth.com Levels] are offering personalized dietary recommendations based on the measurement of an individuals’s [[metabolic flexibility]].

Similar to non-human primates, the effects of CR on lifespan remain controversial in humans. However, what seems clear from obesity studies is that eating too much results in poor health and decreased longevity.<ref>Pifferi, F., Terrien, J., Marchal, J., Dal-Pan, A., Djelti, F., Hardy, I., Chahory, S., Cordonnier, N., Desquilbet, L., Hurion, M. and Zahariev, A., 2018. Caloric restriction increases lifespan but affects brain integrity in grey mouse lemur primates. ''Communications biology'', ''1''(1), pp.1-8.</ref>
<references />
[[Category:Longevity]]

Protein restriction

2022-08-10T02:08:01Z

Rapacurious:

Reduced intake of energy ([[calorie restriction]]) has a long history, but for almost as long scientists have been interested in understanding if restriction of specific types of macronutrients would recapitulate the effects of CR. The results of many early studies were mixed, likely due to differences in dietary protein quality and the degree of restriction.<ref>Green CL, Lamming DW. Regulation of metabolic health by essential dietary amino acids. ''Mech Ageing Dev.'' 2019 Jan;177:186-200. doi:[https://doi.org/10.1016%2Fj.mad.2018.07.004 10.1016/j.mad.2018.07.004]. [Epub 2018 Jul 22]. PMID: 30044947; PMCID: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333505/ PMC6333505]</ref> However, since the end of the last century, interest in protein restriction (PR) as an intervention has been rekindled by studies which have shown that in flies and mice, total protein restriction or restriction of specific essential amino acids can extend lifespan independently of calorie intake.<ref>Mair W, Piper MD, Partridge L. PLoS Biology. 2005 Jul; 3(7):e223. doi: [https://doi.org/10.1371/journal.pbio.0030223 10.1371/journal.pbio.0030223]. Epub 2005 May 31.</ref><ref>Solon-Biet SM, McMahon AC, Ballard JW, Ruohonen K, Wu LE, Cogger VC, Warren A, Huang X, Pichaud N, Melvin RG, Gokarn R, Khalil M, Turner N, Cooney GJ, Sinclair DA, Raubenheimer D, Le Couteur DG, Simpson SJ. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab. 2014 Mar 4; 19(3):418-30. doi: [https://doi.org/10.1016/j.cmet.2014.02.009 10.1016/j.cmet.2014.02.009]. </ref>

== Human studies ==
A number of long-term studies suggest that lower protein diets are associated

== Animal Studies ==

<references />
[[Category:Draft]]
[[Category:Longevity]]

Protein restriction

2022-08-09T19:20:16Z

Rapacurious: article creation more to come

Reduced intake of energy ([[calorie restriction]]) has a long history, but for almost as long scientists have been interested in understanding if restriction of specific types of macronutrients would recapitulate the effects of CR. The results of many early studies were mixed, likely due to differences in dietary protein quality and the degree of restriction.<ref>Green CL, Lamming DW. Regulation of metabolic health by essential dietary amino acids. ''Mech Ageing Dev.'' 2019 Jan;177:186-200. doi:[https://doi.org/10.1016%2Fj.mad.2018.07.004 10.1016/j.mad.2018.07.004]. [Epub 2018 Jul 22]. PMID: 30044947; PMCID: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333505/ PMC6333505]</ref>

However, since the end of the last century, interest in protein restriction (PR) as an intervention has been rekindled by studies which have shown that in flies and mice, total protein restriction or restriction of specific essential amino acids can extend lifespan independently of calorie intake.<ref>Mair W, Piper MD, Partridge L. PLoS Biology. 2005 Jul; 3(7):e223. doi: [https://doi.org/10.1371/journal.pbio.0030223 10.1371/journal.pbio.0030223]. Epub 2005 May 31.</ref><ref>Solon-Biet SM, McMahon AC, Ballard JW, Ruohonen K, Wu LE, Cogger VC, Warren A, Huang X, Pichaud N, Melvin RG, Gokarn R, Khalil M, Turner N, Cooney GJ, Sinclair DA, Raubenheimer D, Le Couteur DG, Simpson SJ. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab. 2014 Mar 4; 19(3):418-30. doi: [https://doi.org/10.1016/j.cmet.2014.02.009 10.1016/j.cmet.2014.02.009]. </ref>

<references />
[[Category:Draft]]
[[Category:Longevity]]

Rapamycin

2022-08-09T19:09:44Z

Rapacurious: font fix

[[File:Rapamycin2.jpg|thumb|211x211px|The chemical structure of rapamycin.]]
Rapamycin, also known by its brand name Rapamune®, is a compound used to prevent the rejection of organ transplants by the immune system. Rapamycin is a natural antifungal produced by soil bacteria of Eastern Island, named after its native island, Rapa Nui.<ref>''Sirolimus - Wikipedia''. En.wikipedia.org. (2021). Retrieved 27 May 2021, from <nowiki>https://en.wikipedia.org/wiki/Sirolimus</nowiki>.</ref>

Rapamycin was approved by the US Food and Drug Administration in September 1999 and is marketed under the trade name Rapamune® by Pfizer.<ref>Accessdata.fda.gov. 2021. ''Drug Approval Package: Rapamune (Sirolimus) NDA# 021083''. [online] Available at: <<nowiki>https://www.accessdata.fda.gov/drugsatfda_docs/nda/99/21083A.cfm</nowiki>> [Accessed 27 May 2021].</ref> At a high dose, rapamycin has an immunosuppressant function that is used in preventing rejection of kidney transplants by the immune system. It is also used to coat coronary stents, and to treat rare lung diseases.<ref>Doggrell, S. (2006). Sirolimus- or paclitaxel-eluting stents for coronary artery revascularisation. ''Expert Opinion On Pharmacotherapy'', ''7''(2), 225-228. <nowiki>https://doi.org/10.1517/14656566.7.2.225</nowiki></ref> The patent on rapamycin has expired, and pharmacological companies have developed similar drugs such as everolimus.<ref>Cancer, C. (2021). ''Everolimus - Drug Information - Chemocare''. Chemocare.com. Retrieved 27 May 2021, from <nowiki>http://chemocare.com/chemotherapy/drug-info/everolimus.aspx</nowiki>.</ref>

Rapamycin has been shown to extend healthy lifespan in worms, yeast, flies, and mice.<ref>[https://science.sciencemag.org/content/328/5976/321.abstract?casa_token=ptqabKjuDxMAAAAA:qBvrJQ01NpHQ8WyJSQf6HeQIhaaexgZXZMvi9i_AP4fE2iHhvMzLV-z3_eF5T3rEauJkg0eQNQFfbA Fontana, L., Partridge, L., & Longo, V. D. (2010). Extending healthy life span—from yeast to humans. ''science'', ''328''(5976), 321-326.]</ref> Some physicians and scientists have thus suggested that rapamycin may slow down aging.<ref name=":2" /><ref>Johnson, S. C., Rabinovitch, P. S., & Kaeberlein, M. (2013). mTOR is a key modulator of ageing and age-related disease. ''Nature'', ''493''(7432), 338-345.</ref>

Scientists who study the biology of aging believe that, due to evidence of rapamycin slowing aging in animals, it could be used to prevent the onset of all age-related diseases in humans – to become one of the first identified “anti-aging drugs”.<ref name=":2">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814615/ Blagosklonny, M. (2019). Rapamycin for longevity: opinion article. ''Aging'', ''11''(19), 8048-8067. https://doi.org/10.18632/aging.102355]</ref><ref>Blagosklonny, M. (2006). Aging and Immortality: Quasi-Programmed Senescence and Its Pharmacologic Inhibition. ''Cell Cycle'', ''5''(18), 2087-2102. <nowiki>https://doi.org/10.4161/cc.5.18.3288</nowiki></ref> It is being repurposed for humans as an anti-aging drug, such as in the PEARL clinical study, which is expected to conclude in 2023.<ref>[https://clinicaltrials.gov/ct2/show/NCT04488601 ''Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity Study - Full Text View - ClinicalTrials.gov''. Clinicaltrials.gov. (2021). Retrieved 27 May 2021, from https://clinicaltrials.gov/ct2/show/NCT04488601.]</ref>

== Evidence of increased healthspan or lifespan ==

=== Dogs ===
There is preliminary evidence that rapamycin may prevent age-related decline in dogs. One study showed statistically significant improvements in heart function in dogs receiving rapamycin, relative to those that received placebo, similar to what has been observed in older laboratory mice.<ref name=":9">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411365/ Urfer, S. R., Kaeberlein, T. L., Mailheau, S., Bergman, P. J., Creevy, K. E., Promislow, D. E., & Kaeberlein, M. (2017). A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs. ''Geroscience'', ''39''(2), 117-127.]</ref> As part of the Dog Aging Project at the University of Washington, the TRIAD study is testing whether rapamycin can extend healthy lifespan in pet dogs.
=== Mice ===
In multiple studies in different breeds of mice, rapamycin demonstrates a robust effect on increasing healthy lifespan. Rapamycin significantly extends lifespan in approximately 90% of the mice models it has been tested in.<ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.</ref>

In 2009, rapamycin was shown to increase the lifespan of both male and female mice when given in late life (600 days).<ref name=":5">[https://doi.org/10.1038%2Fnature08221 Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., ... & Miller, R. A. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. ''nature'', ''460''(7253), 392-395.]</ref> Mean survival was extended by 28% for males and 38% for females, while maximal lifespan increased by 9% for males and 14% for females.<ref name=":5" /> This was the first evidence that the lifespan of a mammal could be significantly increased by a pharmacological drug. This mouse study is special because the results were obtained following the US National Institute on Aging's Interventions Testing Program (ITP) protocol. The ITP is regarded as the gold standard for testing drugs that target aging.<ref>Nadon, N. L., Strong, R., Miller, R. A., Nelson, J., Javors, M., Sharp, Z. D., ... & Harrison, D. E. (2008). Design of aging intervention studies: the NIA interventions testing program. ''Age'', ''30''(4), 187-199.</ref>

The landmark 2009 study also showed that rapamycin could increase healthy lifespan when given in old age. This has important implications for human testing, as it suggests that the drug might still exhibit healthspan and lifespan benefits even when given to the elderly. Rapamycin contrasts with calorie restriction in this regard; some evidence suggests that calorie restriction needs to be practiced from early adulthood, and may even fail to provide benefit for animals that are already old.<ref>Szafranski, K., & Mekhail, K. (2014). The fine line between lifespan extension and shortening in response to caloric restriction. ''Nucleus'', ''5''(1), 56-65.</ref>
==== Rapidly aging mice models ====
Using a mouse model that mimics the accelerated aging disease Hutchinson-Gilford progeria, rapamycin was shown to increase lifespan by over 50%. It also improved cardiac and skeletal muscle function in these mice.<ref>[https://doi.org/10.1126%2Fscitranslmed.3003802 Ramos, F. J., Chen, S. C., Garelick, M. G., Dai, D. F., Liao, C. Y., Schreiber, K. H., ... & Kennedy, B. K. (2012). Rapamycin reverses elevated mTORC1 signaling in lamin A/C–deficient mice, rescues cardiac and skeletal muscle function, and extends survival. ''Science translational medicine'', ''4''(144), 144ra103-144ra103.]
</ref> In one short-lived mutant strain of mice that mimics Leigh syndrome, rapamycin was shown to extend maximum life span nearly three-fold.<ref>[https://www.ncbi.nlm.nih.gov/pubmed/24231806/ Johnson, S. C., Yanos, M. E., Kayser, E. B., Quintana, A., Sangesland, M., Castanza, A., ... & Kaeberlein, M. (2013). mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome. ''Science'', ''342''(6165), 1524-1528.]</ref>

==== Middle-aged mice ====
Several recent studies have shown that rapamycin can extend the lifespan of middle-aged or aged mice. One study showed that treating 20-month-old mice (the equivalent of 56–69 years in humans) with rapamycin for only 3 months resulted in a dramatic increase in median lifespan of up to 60%.<ref>Bitto, A., Ito, T.K., Pineda, V.V., LeTexier, N.J., Huang, H.Z., Sutlief, E., Tung, H., Vizzini, N., Chen, B., Smith, K. and Meza, D., 2016. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. ''elife'', ''5'', p.e16351.</ref> A study from 2020 showed that administering rapamycin in late life enhanced the lifespan of male but not female mice, providing evidence for sex differences in rapamycin response.<ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13269 Strong, R., Miller, R. A., Bogue, M., Fernandez, E., Javors, M. A., Libert, S., ... & Harrison, D. E. (2020). Rapamycin‐mediated mouse lifespan extension: Late‐life dosage regimes with sex‐specific effects. ''Aging cell'', ''19''(11), e13269.]</ref> Aged female mice administered rapamycin once every 5 days starting at 20 months of age also extended lifespan.<ref>Arriola Apelo SI, Pumper CP, Baar EL, Cummings NE, Lamming DW. Intermittent administration of rapamycin extends the lifespan of female C57BL/6J mice. ''J Gerontol A Biol Sci Med Sci''. 2016 Jul; 71(7):876-81. doi: [https://academic.oup.com/biomedgerontology/article/71/7/876/2605199 10.1093/gerona/glw064]. Epub 2016 Apr 18. PMID: 27091134; PMCID: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906329/ PMC4906329]</ref> These studies were important as they suggest that much of the health and longevity benefits of rapamycin could be achieved even when dosed in late life or intermittently, as opposed to only being effective with continual dosing in early life.

=== Yeast ===
Inhibition of TOR signalling by rapamycin significantly increases the lifespan of yeast known as ''Saccharomyces cerevisiae.''<ref>Powers, R. W., Kaeberlein, M., Caldwell, S. D., Kennedy, B. K., & Fields, S. (2006). Extension of chronological life span in yeast by decreased TOR pathway signaling. ''Genes & development'', ''20''(2), 174-184.</ref>

=== Flies ===
Rapamycin extends the lifespan of the fruitfly, ''Drosophila melanogaster.'' The extent of lifespan extension observed is beyond what is achievable by flies undergoing other pro-longevity interventions like dietary restriction, or in mutant flies with mild decrements in insulin/insulin-like growth factor signaling (IIS).<ref>Bjedov, I., Toivonen, J. M., Kerr, F., Slack, C., Jacobson, J., Foley, A., & Partridge, L. (2010). Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. ''Cell metabolism'', ''11''(1), 35-46.</ref> Combining rapamycin with two other drugs that target metabolic pathways, lithium and trametinib, results in additive lifespan extension effects, substantially increasing ''Drosophila'' lifepsan by 48%.<ref>Castillo-Quan, J. I., Tain, L. S., Kinghorn, K. J., Li, L., Grönke, S., Hinze, Y., ... & Partridge, L. (2019). A triple drug combination targeting components of the nutrient-sensing network maximizes longevity. ''Proceedings of the National Academy of Sciences'', ''116''(42), 20817-20819.</ref>

=== Roundworms ===
TOR inhibition by rapamycin extends lifespan in ''Caenorhabditis elegans,'' a roundworm nematode widely used in research areas of the biology of aging. The beneficial effects of rapamycin in ''C. elegans'' seem to be mediated via the SKN-1/Nrf and DAF-16/FoxO pathways.<ref>Robida-Stubbs, S., Glover-Cutter, K., Lamming, D. W., Mizunuma, M., Narasimhan, S. D., Neumann-Haefelin, E., ... & Blackwell, T. K. (2012). TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. ''Cell metabolism'', ''15''(5), 713-724.</ref>

=== Age-related diseases ===
Rapamycin has been investigated in specific diseases, showing major impacts on reducing mouse cancer risk, cardiac diseases, neurodegenerative-like processes, and many other pathologies.<ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.
</ref>

===== Cancer =====
In the transgenic HER-2/neu mouse model, mice die prematurely due to susceptibility to cancer.<ref name=":6" /> Rapamycin was hypothesized to improve survival in this model due to its ability to slow aging, which would also address an age-related disease like cancer.<ref name=":6" /> The drug was shown to extend maximal lifespan, by delaying aging in multiple different organs and also suppressing cancer development.<ref name=":6">Anisimov, V. N., Zabezhinski, M. A., Popovich, I. G., Piskunova, T. S., Semenchenko, A. V., Tyndyk, M. L., ... & Blagosklonny, M. V. (2010). Rapamycin extends maximal lifespan in cancer-prone mice. ''The American journal of pathology'', ''176''(5), 2092-2097.</ref>

Other studies suggest that rapamycin can extend lifespan in mouse models where cancer naturally develops, such as in mice prone to cancer due to the Apc tumor suppressor gene mutation, or in mice heterozygous for the Rb1 tumor supressor gene, among others.<ref>Hasty, P., Livi, C. B., Dodds, S. G., Jones, D., Strong, R., Javors, M., ... & Sharp, Z. D. (2014). eRapa restores a normal life span in a FAP mouse model. ''Cancer Prevention Research'', ''7''(1), 169-178.</ref><ref>Livi, C. B., Hardman, R. L., Christy, B. A., Dodds, S. G., Jones, D., Williams, C., ... & Sharp, Z. D. (2013). Rapamycin extends life span of Rb1+/− mice by inhibiting neuroendocrine tumors. ''Aging (Albany NY)'', ''5''(2), 100.</ref><ref>Hambright, H. G., Hurez, V., & Curiel, T. J. (2020). Chronic Mechanistic Target of Rapamycin Inhibition: Preventing Cancer to Delay Aging or Vice Versa?. ''Geriatric Oncology'', 111-128.</ref>

A group of investigators in Germany have argued, based on their experiment in C57BL/6J Rj inbred mice, that rapamycin extends lifespan mainly through delaying cancer incidence, instead of via slowed aging.<ref>Neff, F., Flores-Dominguez, D., Ryan, D. P., Horsch, M., Schröder, S., Adler, T., ... & Ehninger, D. (2013). Rapamycin extends murine lifespan but has limited effects on aging. ''The Journal of clinical investigation'', ''123''(8), 3272-3291.</ref> A further analysis of the paper by Johnson et al. pointed to several important limitations of the study, suggesting that such a conclusion may be premature.<ref name=":82">Johnson, S. C., Martin, G. M., Rabinovitch, P. S., & Kaeberlein, M. (2013). Preserving youth: does rapamycin deliver? ''Science translational medicine'', ''5''(211), 211fs40.</ref> Key limitations included the lack of dose-response profiling of rapamycin; studying only the male sex, which is known to respond less to rapamycin likely in part due to sex differences in drug metabolism; lack of reporting on tumor size and incidence, required to determine whether lifespan extension occured only via slowed cancer or from a general effect on aging; and, the cross-sectional nature of the study, which would have reduced sensitivity for detecting age-related organ/tissue changes compared to longitudinal assays.<ref name=":82" /><ref>Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref>Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much? ''Journal of genetics and genomics,'' ''41''(9), 459.</ref>

===== Heart Disease =====
One study investigated the effects of late-life rapamycin dosing in aged female mice, observing a reversal of age-related heart dysfunction.<ref name=":16">Flynn, J. M., O'Leary, M. N., Zambataro, C. A., Academia, E. C., Presley, M. P., Garrett, B. J., ... & Melov, S. (2013). Late‐life rapamycin treatment reverses age‐related heart dysfunction. ''Aging cell'', ''12''(5), 851-862.

</ref> This included benefits to ejection fraction, cardiac hormones, and reduced inflammation, although no effect was observed for heart fibrosis.<ref name=":16" /> Additionally, improvements in behaviour and physical function were demonstrated.<ref name=":16" /> Another study in naturally aged mice showed improvements in cardiac muscle stiffness, diastolic function with rapamycin.<ref name=":7">Quarles, E., Basisty, N., Chiao, Y. A., Merrihew, G., Gu, H., Sweetwyne, M. T., ... & Rabinovitch, P. S. (2020). Rapamycin persistently improves cardiac function in aged, male and female mice, even following cessation of treatment. ''Aging Cell'', ''19''(2), e13086.</ref> Improvements in heart function were shown with only a brief treatment course of 8 weeks.<ref name=":7" /> Benefits persisted even after rapamycin was stopped, which appears consistent with the hypothesis that rapamycin slows aging.<ref name=":7" />

===== Alzheimer's =====
Alzheimer's Disease (AD) is a progressive neurodegenerative disease for which age is the greatest risk factor.<ref>https://www.science.org/doi/10.1126/scitranslmed.aar4289</ref> In an Alzheimer's model of transgenic PDAPP mice, rapamycin was shown to reduce Amyloid-β, one of the hallmarks of AD.<ref name=":8" /> This led to alleviation of AD-like symptoms, such as restored cognition and memory.<ref name=":8">Spilman, P., Podlutskaya, N., Hart, M. J., Debnath, J., Gorostiza, O., Bredesen, D., ... & Galvan, V. (2010). Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-β levels in a mouse model of Alzheimer's disease. ''PloS one'', ''5''(4), e9979.</ref> Similarly, another major AD hallmark known as tau was mitigated by rapamycin in a tauopathy mouse model.<ref name=":14">Ozcelik, S., Fraser, G., Castets, P., Schaeffer, V., Skachokova, Z., Breu, K., ... & Winkler, D. T. (2013). Rapamycin attenuates the progression of tau pathology in P301S tau transgenic mice. ''PloS one'', ''8''(5), e62459.</ref> The mechanism of clearance of these proteins was linked to autophagy, with benefits seen regardless of whether it was dosed early for prevention, or in late life as treatment.<ref name=":14" />

=== Dose response ===
Rapamycin has shown a dose-response in which higher doses produce larger lifespan extension effects. UMHET3 mice of diverse genetic background were treated with varying doses of dietary rapamycin at 4.7, 14, or 42 ppm, revealing that those fed with the highest rapamycin dose had the greatest lifespan extension.<ref>Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref name=":0">[[Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much?. Journal of genetics and genomics= Yi chuan xue bao, 41(9), 459.|Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much? ''Journal of genetics and genomics,'' ''41''(9), 459.]]</ref> Sex differences in response to rapamycin have been hypothesized to also be related to the effective dose, due to male/female differences in drug metabolism. The optimal dose for longevity in mice remains to be seen, but determining this dose will require consideration of the side effect profile of rapamycin.<ref name=":0" />

== Mechanism ==

=== Manipulating metabolic pathways - differences to calorie restriction ===
Rapamycin has often been described as a 'calorie restriction (CR) mimetic'.<ref name=":15">Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref name=":4" /> This is in part because CR also inhibits the nutrient-sensing mammalian target of rapamycin (mTOR) pathway.<ref name=":3">Cornu, M., Albert, V., & Hall, M. N. (2013). mTOR in aging, metabolism, and cancer. ''Current opinion in genetics & development'', ''23''(1), 53-62.</ref> mTOR plays key roles in cellular growth in response to amino acids, including effects that inhibit cancer and aging mechanisms.<ref name=":3" /><ref>Johnson, S. C., Rabinovitch, P. S., & Kaeberlein, M. (2013). mTOR is a key modulator of ageing and age-related disease. ''Nature'', ''493''(7432), 338-345.</ref> However, later studies have disentangled the effects of rapamycin from that of caloric restriction, showing that they differ significantly.<ref name=":15" /><ref name=":4">Unnikrishnan, A., Kurup, K., Salmon, A. B., & Richardson, A. (2020). Is rapamycin a dietary restriction mimetic?. ''The Journals of Gerontology: Series A'', ''75''(1), 4-13.</ref> For example, unlike 5 months of CR, rapamycin does not decrease leptin, insulin, IGF-1, or FGF-21 in genetically diverse UM-HET3 mice.<ref name=":15" /> This has important implications for understanding biological aging, including the possibility of using CR and rapalogs in combination therapy to slow aging.<ref name=":4" />

Though distinct from CR, fasting inhibits muscle-specific mTOR signaling with reduced effect in old vs young mice, indicating a poorer autophagy and proteosomal degradation response with age.<ref>White, Z., White, R. B., McMahon, C., Grounds, M. D., & Shavlakadze, T. (2016). High mTORC1 signaling is maintained, while protein degradation pathways are perturbed in old murine skeletal muscles in the fasted state. ''The international journal of biochemistry & cell biology'', ''78'', 10-21.</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/21179166/ Sengupta, S., Peterson, T. R., Laplante, M., Oh, S., & Sabatini, D. M. (2010). mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. ''Nature'', ''468''(7327), 1100-1104.]</ref> However, the ability for rapamycin to inhibit mTOR appears to remain robust throughout life, and significant extension of median and maximal lifespan can be achieved even when treatment is initiated in mid-to-late life.<ref name=":5" /><ref>Blagosklonny, M. V. (2019). Rapamycin for longevity: opinion article. ''Aging (Albany NY)'', ''11''(19), 8048.
</ref> Rapamycin contrasts significantly with CR and fasting, in that the latter could be detrimental when used in late life.<ref>Kemnitz, J. W. (2011). Calorie restriction and aging in nonhuman primates. ''Ilar Journal'', ''52''(1), 66-77.</ref><ref>Goto, S., Takahashi, R., Radak, Z., & Sharma, R. (2007). Beneficial biochemical outcomes of late‐onset dietary restriction in rodents. ''Annals of the New York Academy of Sciences'', ''1100''(1), 431-441.</ref><ref>Kristan, D. M. (2008). Calorie restriction and susceptibility to intact pathogens. ''Age'', ''30''(2), 147-156.</ref> Rapamycin also targets multiple diseases of aging, but seemingly in a segmented, tissue-specific manner.<ref>Neff, F., Flores-Dominguez, D., Ryan, D. P., Horsch, M., Schröder, S., Adler, T., ... & Ehninger, D. (2013). Rapamycin extends murine lifespan but has limited effects on aging. ''The Journal of clinical investigation'', ''123''(8), 3272-3291.</ref>

=== mTORC1 and mTORC2 ===
In non-mammals the mTOR equivalent is known as the target of rapamycin (TOR), first discovered by a team led by Michael Hall in the yeast ''Saccharomyces cerevisiae''.<ref>Thomas, G., & Hall, M. N. (1997). TOR signalling and control of cell growth. ''Current opinion in cell biology'', ''9''(6), 782-787.</ref><ref>Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., & Hall, M. N. (1993). Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. ''Cell'', ''73''(3), 585-596.</ref> mTOR signalling appears to be evolutionarily conserved, and this extends further to include various mammals, such as mice, rats, and dogs.

Rapamycin acts on mTOR, with multiple signaling functions subdivided across two major protein complexes known as mTORC1 and mTORC2.<ref name=":3" /> There is some evidence suggesting that the health and lifespan benefit of rapamycin is more related to inhibition of mTORC1 than mTORC2.<ref name=":19">Lamming, D. W., Ye, L., Katajisto, P., Goncalves, M. D., Saitoh, M., Stevens, D. M., ... & Baur, J. A. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. ''science'', ''335''(6076), 1638-1643.</ref><ref name=":11">Lamming, D. W., Mihaylova, M. M., Katajisto, P., Baar, E. L., Yilmaz, O. H., Hutchins, A., ... & Sabatini, D. M. (2014). Depletion of Rictor, an essential protein component of m TORC 2, decreases male lifespan. ''Aging cell'', ''13''(5), 911-917.</ref> In mice, males exhibit weaker lifespan extension effects from rapamycin than in females. One study suggests that inhibiting mTORC2 explains why the sex difference in the response to mTOR inhibition by rapamycin.<ref name=":11" />

=== Effects on glucoregulatory control ===
A noted issue regarding rapamycin is the disruption of glucose metabolism with chronic dosing, which has been observed in both humans and mice. In mice, this side effect has been shown to be due in part to disruption of mTORC2 in the liver, leading to hepatic insulin resistance.<ref name=":19" /> This effect has previously been shown to be reversible upon stopping the drug in both lean and obese mice.<ref name=":18">Liu, Y., Diaz, V., Fernandez, E., Strong, R., Ye, L., Baur, J. A., ... & Salmon, A. B. (2014). Rapamycin-induced metabolic defects are reversible in both lean and obese mice. ''Aging (Albany NY)'', ''6''(9), 742.</ref> Whether disrupted glucose metabolism is dispensable for the lifespan extending effects of rapamycin remains controversial.<ref>Blagosklonny, M. V. (2012). Once again on rapamycin-induced insulin resistance and longevity: despite of or owing to. ''Aging (Albany NY)'', ''4''(5), 350.</ref><ref>[https://www.nature.com/articles/s41419-019-1822-8 Blagosklonny, M. V. (2019). Fasting and rapamycin: diabetes versus benevolent glucose intolerance. ''Cell death & disease'', ''10''(8), 1-10.]</ref><ref>[https://www.science.org/doi/full/10.1126/science.1215135 Lamming, D. W., Ye, L., Katajisto, P., Goncalves, M. D., Saitoh, M., Stevens, D. M., ... & Baur, J. A. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. ''science'', ''335''(6076), 1638-1643.]</ref><ref>Strong, R., Miller, R. A., Antebi, A., Astle, C. M., Bogue, M., Denzel, M. S., ... & Harrison, D. E. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α‐glucosidase inhibitor or a Nrf2‐inducer. ''Aging cell'', ''15''(5), 872-884.</ref> Rapamycin has previously been shown to increase insulin sensitivity with acute dosing, while decreasing insulin sensitivity with chronic dosing.<ref>Ye, L., Varamini, B., Lamming, D. W., Sabatini, D. M., & Baur, J. A. (2012). Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2. ''Frontiers in genetics'', ''3'', 177.</ref>

=== Reducing the effects of cellular senescence ===
The accumulation of senescent cells is thought to be an important mechanism underlying aging. Rapamycin is regarded as a senomorphic that may inhibit the pro-inflammatory secretory phenotype produced by senescent cells in humans, mice, and rats.<ref>[[Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., ... & Perez, V. I. (2017). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism. Aging cell, 16(3), 564-574.|Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., ... & Perez, V. I. (2017). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism. ''Aging cell'', ''16''(3), 564-574.]]</ref><ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.
</ref> A preliminary study in humans aged 40 years or older showed that topical rapamycin reduced markers of cellular senescence in the skin and improved its physical appearance.<ref>[https://doi.org/10.1007%2Fs11357-019-00113-y Chung, C. L., Lawrence, I., Hoffman, M., Elgindi, D., Nadhan, K., Potnis, M., ... & Sell, C. (2019). Topical rapamycin reduces markers of senescence and aging in human skin: an exploratory, prospective, randomized trial. ''Geroscience'', ''41''(6), 861-869.]
</ref>

== Human clinical trials ==
[[File:Rapamycin.jpg|thumb|235x235px|Part of the rationale of the PEARL study is to determine the optimal dose of rapamycin to potentially slow aging.<ref name=":2" />]]

=== PEARL study ===
Rapamycin is currently being tested for safety and efficacy in a clinical trial called the Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity (PEARL) study. The clinical trial aims to systematically investigate the use of rapamycin to promote healthy longevity, and is expected to conclude in 2023.<ref>[https://clinicaltrials.gov/ct2/show/NCT04488601 ''Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity Study - Full Text View - ClinicalTrials.gov''. Clinicaltrials.gov. (2021). Retrieved 27 May 2021, from https://clinicaltrials.gov/ct2/show/NCT04488601.]</ref>

The study will begin with 200 adults aged 50 years or older who will receive rapamycin for up to one year. The study is being conducted by AgelessRx, a new company dedicated to developing scientifically supported interventions to prevent and treat age-related diseases, in collaboration with the University of California.<ref>[https://www.longevity.technology/agelessrx-and-funding-an-irb-approved-rapamycin-trial/ ''AgelessRx and funding an IRB-approved rapamycin trial - Longevity.Technology''. Longevity.Technology. (2021). Retrieved 27 May 2021, from https://www.longevity.technology/agelessrx-and-funding-an-irb-approved-rapamycin-trial/.]</ref>

The trial aims to obtain clinical data at 6 and 12 months of treatment, such as via testing of blood, body composition DXA, fecal microbiome, immune function, inflammation, skeletal muscle, and epigenetic aging clocks.

== Dog clinical trials ==
The Dog Aging Project is a US Government NIH-funded initiative investigating dog aging.<ref>https://dogagingproject.org/</ref> The project is led by Professor Matt Kaeberlein at the University of Washington.<ref name=":10">Kaeberlein, M., Creevy, K. E., & Promislow, D. E. (2016). The dog aging project: translational geroscience in companion animals. ''Mammalian genome'', ''27''(7), 279-288.</ref>

The Test of Rapamycin In Aging Dogs (TRIAD) study is investigating rapamycin as a treatment to slow aging in dogs. The investigators hope to increase healthy canine lifespan with rapamycin by delaying the onset of age-related diseases like cancer and heart disease.<ref name=":9" /><ref name=":10" />

Aging biology scientists believe that studying dog aging might not only help improve canine healthspan, but also have implications for humans.<ref name=":10" /> Dogs may be a useful animal model because they share the same environment that humans live in, and suffer from similar chronic diseases with aging.<ref>Hoffman, J. M., Creevy, K. E., Franks, A., O'Neill, D. G., & Promislow, D. E. (2018). The companion dog as a model for human aging and mortality. ''Aging Cell'', ''17''(3), e12737.</ref>

===== Heart disease =====
One randomized-controlled trial in 24 middle-aged dogs treated with low-dose rapamycin showed suggestion of partial reversal of age-related heart dysfunction, as measured via echocardiography. The intervention was well-tolerated, with no clinically meaningful adverse events noted with a non-immunosuppressive dose of rapamycin during the 10 week period. This was a small study over a relatively short duration; further testing in larger clinical studies will be necessary to determine whether rapamycin can be used to treat age-related heart disease in dogs.

== Regulatory approval ==
Rapamycin was approved by the US Food and Drug Administration (FDA) in 1999 to prevent organ rejection in liver transplant patients, and has been marketed under the brand name Rapamune.<ref>[https://link.springer.com/article/10.1007/s11357-020-00274-1 Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.]</ref> The patent on rapamycin has expired, and chemically similar compounds called 'rapalogs' are being researched by biotechnology companies.<ref>[https://pubs.acs.org/doi/full/10.1021/acsmedchemlett.9b00215 Abdel-Magid, A. F. (2019). Rapalogs potential as practical alternatives to rapamycin.]
</ref> It is not currently approved for use as an anti-aging medication, due to lack of human clinical data for this purpose.

== Safety ==
Rapamycin has been used to treat millions of patients over several decades since obtaining FDA approval in 1999. It is generally considered safe in humans, but only when used under clinical supervision for specific indications. Various side effects have been reported with the high dose of rapamycin used to prevent rejection in organ transplant patients, who are often concurrently treated with multiple other medications.<ref>Webster, A. C., Lee, V. W., Chapman, J. R., & Craig, J. C. (2006). Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials. ''Transplantation'', ''81''(9), 1234-1248.</ref> These include pain, headache, fever, high blood pressure, glucose intolerance, nausea, abdominal pain, constipation, diarrhea, thrombocytopenia, leukopenia, among others. However, side effects are mostly reversible and represent worst-case scenarios, particularly because the patients sampled in clinical studies are already severely ill and taking high doses of the drug along with other medications.<ref>Bischof, E., Siow, R.C., Zhavoronkov, A. and Kaeberlein, M., 2021. The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19. ''The Lancet Healthy longevity'', ''2''(2), pp.e105-e111.
</ref>

Case studies of the safety profile of rapamycin in the context of overdosing have suggested that it may have a large margin of safety or a high median lethal dose, but only in the acute setting.<ref>Ceschi, A., Heistermann, E., Gros, S., Reichert, C., Kupferschmidt, H., Banner, N. R., ... & Taegtmeyer, A. B. (2015). Acute sirolimus overdose: a multicenter case series. ''PLoS One'', ''10''(5), e0128033.</ref> The distinction with chronic high dose mTOR inhibition must be made because resultant immunosuppression can lead to susceptibility to infection with fatal consequences. Some preclinical data suggests that the longevity benefits of rapamycin may be retained via intermittent dosing.<ref name=":2" /> In considering the known clinical data about rapamycin's controversial safety at continuous, high doses, some researchers have proposed that rapamycin should dosed intermittently to minimize side effects while sufficiently inhibiting mTOR for an effect on aging.<ref name=":2" />

From a longevity perspective, there is a lack of published clinical data demonstrating the safety of rapamycin in healthy adults. One randomized pilot study of rapamycin in 25 older adults aged 70-95 showed no clinically significant effects, including a lack of effect on immune function.<ref name=":13">Kraig, E., Linehan, L. A., Liang, H., Romo, T. Q., Liu, Q., Wu, Y., ... & Kellogg Jr, D. L. (2018). A randomized control trial to establish the feasibility and safety of rapamycin treatment in an older human cohort: Immunological, physical performance, and cognitive effects. ''Experimental gerontology'', ''105'', 53-69.</ref> This was a small study with a low dose of rapamycin, dosed over a short duration of 8 weeks. Considering the fact that, based on preclinical animal data, any potential benefit of rapamycin for aging will require long-term dosing, further testing in clinical trials is necessary to better characterize safety.<ref name=":13" />

Clinical trials such as the PEARL study are needed to provide evidence for the safety profile of rapamycin in otherwise healthy older adults.

== Rapalogs ==
Rapalogs are molecules with similar mechanism to rapamycin, primarily via mTORC1 inhibition. These drugs are generally predicted to function similarly to rapamycin in enhancing lifespan and reducing age-related decline in physiological function. However, only one rapalog, everolimus, has published clinical data in this context.<ref>[https://www.thelancet.com/servlet/linkout?suffix=e_1_5_1_2_55_2&dbid=8&doi=10.1016/S2666-7568(20)30068-4&key=24379984&cf= Kaeberlein, M. (2013). mTOR inhibition: from aging to autism and beyond. ''Scientifica'', ''2013''.]</ref> RTB101 has also been described as a selective mTOR inhibitor, but some controversy exists.<ref>Kaeberlein, M. (2020). RTB101 and immune function in the elderly: interpreting an unsuccessful clinical trial. ''Translational Medicine of Aging'', ''4'', 32-34.</ref><ref>Bischof, E., Siow, R. C., Zhavoronkov, A., & Kaeberlein, M. (2021). The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19. ''The Lancet Healthy longevity'', ''2''(2), e105-e111.</ref>

=== mTOR inhibition improves immune function in the elderly ===

In a phase 2 randomized clinical trial published in ''Science Translational Medicine'' in 2014, low-dose TORC1 inhibition with the rapalog everolimus showed improvement in immune function in the elderly. The clinical trial enrolled 218 adults aged ≥65 years, observing decreased incidence of all infections, as well as improved influenza vaccination responses and upregulation of antiviral immunity.<ref name=":17">Mannick, J. B., Del Giudice, G., Lattanzi, M., Valiante, N. M., Praestgaard, J., Huang, B., ... & Klickstein, L. B. (2014). mTOR inhibition improves immune function in the elderly. ''Science translational medicine'', ''6''(268), 268ra179-268ra179.</ref>

Everolimus enhanced the influenza vaccine response by approximately 20% at relatively well tolerated doses.<ref name=":17" /> One mechanism was related to a reduction in the percentage of CD4 and CD8 T cells expressing the programmed death-1 receptor, which has increased expression with age and a major role in inhibiting T cell signaling.<ref name=":17" /> These findings suggest that, at an appopriate dose, mTOR inhibition may improve the age-related decline in immune function in the elderly.

=== TORC1 inhibition enhances immune function and reduces infections in the elderly ===

A phase 2a trial clinical trial randomized 264 older adults to treatment with everolimus and placebo, and was published in ''Science Translational Medicine'' in 2018. The trial showed potential for reducing the effects of immune aging, with improvement in influenza vaccination response in the elderly.<ref name=":1">Mannick, J. B., Morris, M., Hockey, H. U. P., Roma, G., Beibel, M., Kulmatycki, K., ... & Klickstein, L. B. (2018). TORC1 inhibition enhances immune function and reduces infections in the elderly. ''Science translational medicine'', ''10''(449).</ref>[[File:Rtb101 Ph2 Ph3.jpg|thumb|461x461px|A) Number of patients with laboratory-confirmed RTIs of ''any severity'' caused by specific viruses, comparing RTB101 group (blue) versus placebo (grey) in the phase 2b, phase 3, and combined phase 2b/3 RCTs B) Number of patients with laboratory-confirmed RTIs with ''severe symptoms'' caused by specific viruses in the RTB101 group (blue) versus placebo (grey) in the phase 2b, phase 3, and combined phase 2b/3 trials. RTI = respiratory tract infection]]

=== Improving immune function in older adults for respiratory tract infections, including coronaviruses ===
Low-dose mTOR inhibition with dactolisib in a Phase 2b and phase 3 trial in the elderly showed reduced coronavirus (non [[COVID-19]]) incidence, as well as reductions in severe symptoms.<ref name=":12">Mannick, J. B., Teo, G., Bernardo, P., Quinn, D., Russell, K., Klickstein, L., ... & Shergill, S. (2021). Targeting the biology of ageing with mTOR inhibitors to improve immune function in older adults: phase 2b and phase 3 randomised trials. ''The Lancet Healthy Longevity'', ''2''(5), e250-e262.</ref> However, the data remains inconclusive as the study was powered statistically for a reduction in clinically symptomatic respiratory tract infections (RTIs), and not laboratory-confirmed RTIs.<ref name=":12" />

Following the success of two phase 2 clinical trials investigating mTOR inhibition for targeting the aging immune system, dactolisib is currently being pursued for the treatment of COVID-19 in a phase 2a placebo-controlled trial (ClinicalTrials.gov Identifier: NCT04584710, NCT04409327), exploring the potential for preventing severe disease in elderly adults with no symptoms, who have been exposed to [[COVID-19]].<ref name=":12" />

Unpublished data from the phase 2 trial of RTB101 for COVID-19 among nursing home patients treated within 3 days from testing positive saw promising results. None of those treated with RTB101 developed symptoms (n=18), while the placebo treated control group had 4 severe cases of disease and 2 deaths. While this was a statistically significant finding, larger trials are warranted for further evidence of potential benefit.

This trial is being run by the biopharmaceutical company resTORbio and has obtained funding from the National Institute on Aging (NIA/NIH). The studies with dactolisib for COVID-19 is one of several clinical trials in the aging biology field aiming to target aging to improve the aging immune system.<ref name=":12" />

== References ==
<references />
[[Category:Pillar Articles]]
[[Category:Drugs]]

Protein restriction

2022-08-09T19:08:01Z

Rapacurious: started page on protein restriction, more to come

Rapamycin

2022-08-09T18:15:59Z

Rapacurious: more descriptions re mTORC2 and side effects, more to come

[[File:Rapamycin2.jpg|thumb|211x211px|The chemical structure of rapamycin.]]
Rapamycin, also known by its brand name Rapamune®, is a compound used to prevent the rejection of organ transplants by the immune system. Rapamycin is a natural antifungal produced by soil bacteria of Eastern Island, named after its native island, Rapa Nui.<ref>''Sirolimus - Wikipedia''. En.wikipedia.org. (2021). Retrieved 27 May 2021, from <nowiki>https://en.wikipedia.org/wiki/Sirolimus</nowiki>.</ref>

Rapamycin was approved by the US Food and Drug Administration in September 1999 and is marketed under the trade name Rapamune® by Pfizer.<ref>Accessdata.fda.gov. 2021. ''Drug Approval Package: Rapamune (Sirolimus) NDA# 021083''. [online] Available at: <<nowiki>https://www.accessdata.fda.gov/drugsatfda_docs/nda/99/21083A.cfm</nowiki>> [Accessed 27 May 2021].</ref> At a high dose, rapamycin has an immunosuppressant function that is used in preventing rejection of kidney transplants by the immune system. It is also used to coat coronary stents, and to treat rare lung diseases.<ref>Doggrell, S. (2006). Sirolimus- or paclitaxel-eluting stents for coronary artery revascularisation. ''Expert Opinion On Pharmacotherapy'', ''7''(2), 225-228. <nowiki>https://doi.org/10.1517/14656566.7.2.225</nowiki></ref> The patent on rapamycin has expired, and pharmacological companies have developed similar drugs such as everolimus.<ref>Cancer, C. (2021). ''Everolimus - Drug Information - Chemocare''. Chemocare.com. Retrieved 27 May 2021, from <nowiki>http://chemocare.com/chemotherapy/drug-info/everolimus.aspx</nowiki>.</ref>

Rapamycin has been shown to extend healthy lifespan in worms, yeast, flies, and mice.<ref>[https://science.sciencemag.org/content/328/5976/321.abstract?casa_token=ptqabKjuDxMAAAAA:qBvrJQ01NpHQ8WyJSQf6HeQIhaaexgZXZMvi9i_AP4fE2iHhvMzLV-z3_eF5T3rEauJkg0eQNQFfbA Fontana, L., Partridge, L., & Longo, V. D. (2010). Extending healthy life span—from yeast to humans. ''science'', ''328''(5976), 321-326.]</ref> Some physicians and scientists have thus suggested that rapamycin may slow down aging.<ref name=":2" /><ref>Johnson, S. C., Rabinovitch, P. S., & Kaeberlein, M. (2013). mTOR is a key modulator of ageing and age-related disease. ''Nature'', ''493''(7432), 338-345.</ref>

Scientists who study the biology of aging believe that, due to evidence of rapamycin slowing aging in animals, it could be used to prevent the onset of all age-related diseases in humans – to become one of the first identified “anti-aging drugs”.<ref name=":2">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814615/ Blagosklonny, M. (2019). Rapamycin for longevity: opinion article. ''Aging'', ''11''(19), 8048-8067. https://doi.org/10.18632/aging.102355]</ref><ref>Blagosklonny, M. (2006). Aging and Immortality: Quasi-Programmed Senescence and Its Pharmacologic Inhibition. ''Cell Cycle'', ''5''(18), 2087-2102. <nowiki>https://doi.org/10.4161/cc.5.18.3288</nowiki></ref> It is being repurposed for humans as an anti-aging drug, such as in the PEARL clinical study, which is expected to conclude in 2023.<ref>[https://clinicaltrials.gov/ct2/show/NCT04488601 ''Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity Study - Full Text View - ClinicalTrials.gov''. Clinicaltrials.gov. (2021). Retrieved 27 May 2021, from https://clinicaltrials.gov/ct2/show/NCT04488601.]</ref>

== Evidence of increased healthspan or lifespan ==

=== Dogs ===
There is preliminary evidence that rapamycin may prevent age-related decline in dogs. One study showed statistically significant improvements in heart function in dogs receiving rapamycin, relative to those that received placebo, similar to what has been observed in older laboratory mice.<ref name=":9">[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411365/ Urfer, S. R., Kaeberlein, T. L., Mailheau, S., Bergman, P. J., Creevy, K. E., Promislow, D. E., & Kaeberlein, M. (2017). A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs. ''Geroscience'', ''39''(2), 117-127.]</ref> As part of the Dog Aging Project at the University of Washington, the TRIAD study is testing whether rapamycin can extend healthy lifespan in pet dogs.
=== Mice ===
In multiple studies in different breeds of mice, rapamycin demonstrates a robust effect on increasing healthy lifespan. Rapamycin significantly extends lifespan in approximately 90% of the mice models it has been tested in.<ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.</ref>

In 2009, rapamycin was shown to increase the lifespan of both male and female mice when given in late life (600 days).<ref name=":5">[https://doi.org/10.1038%2Fnature08221 Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., ... & Miller, R. A. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. ''nature'', ''460''(7253), 392-395.]</ref> Mean survival was extended by 28% for males and 38% for females, while maximal lifespan increased by 9% for males and 14% for females.<ref name=":5" /> This was the first evidence that the lifespan of a mammal could be significantly increased by a pharmacological drug. This mouse study is special because the results were obtained following the US National Institute on Aging's Interventions Testing Program (ITP) protocol. The ITP is regarded as the gold standard for testing drugs that target aging.<ref>Nadon, N. L., Strong, R., Miller, R. A., Nelson, J., Javors, M., Sharp, Z. D., ... & Harrison, D. E. (2008). Design of aging intervention studies: the NIA interventions testing program. ''Age'', ''30''(4), 187-199.</ref>

The landmark 2009 study also showed that rapamycin could increase healthy lifespan when given in old age. This has important implications for human testing, as it suggests that the drug might still exhibit healthspan and lifespan benefits even when given to the elderly. Rapamycin contrasts with calorie restriction in this regard; some evidence suggests that calorie restriction needs to be practiced from early adulthood, and may even fail to provide benefit for animals that are already old.<ref>Szafranski, K., & Mekhail, K. (2014). The fine line between lifespan extension and shortening in response to caloric restriction. ''Nucleus'', ''5''(1), 56-65.</ref>
==== Rapidly aging mice models ====
Using a mouse model that mimics the accelerated aging disease Hutchinson-Gilford progeria, rapamycin was shown to increase lifespan by over 50%. It also improved cardiac and skeletal muscle function in these mice.<ref>[https://doi.org/10.1126%2Fscitranslmed.3003802 Ramos, F. J., Chen, S. C., Garelick, M. G., Dai, D. F., Liao, C. Y., Schreiber, K. H., ... & Kennedy, B. K. (2012). Rapamycin reverses elevated mTORC1 signaling in lamin A/C–deficient mice, rescues cardiac and skeletal muscle function, and extends survival. ''Science translational medicine'', ''4''(144), 144ra103-144ra103.]
</ref> In one short-lived mutant strain of mice that mimics Leigh syndrome, rapamycin was shown to extend maximum life span nearly three-fold.<ref>[https://www.ncbi.nlm.nih.gov/pubmed/24231806/ Johnson, S. C., Yanos, M. E., Kayser, E. B., Quintana, A., Sangesland, M., Castanza, A., ... & Kaeberlein, M. (2013). mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome. ''Science'', ''342''(6165), 1524-1528.]</ref>

==== Middle-aged mice ====
Several recent studies have shown that rapamycin can extend the lifespan of middle-aged or aged mice. One study showed that treating 20-month-old mice (the equivalent of 56–69 years in humans) with rapamycin for only 3 months resulted in a dramatic increase in median lifespan of up to 60%.<ref>Bitto, A., Ito, T.K., Pineda, V.V., LeTexier, N.J., Huang, H.Z., Sutlief, E., Tung, H., Vizzini, N., Chen, B., Smith, K. and Meza, D., 2016. Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice. ''elife'', ''5'', p.e16351.</ref> A study from 2020 showed that administering rapamycin in late life enhanced the lifespan of male but not female mice, providing evidence for sex differences in rapamycin response.<ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13269 Strong, R., Miller, R. A., Bogue, M., Fernandez, E., Javors, M. A., Libert, S., ... & Harrison, D. E. (2020). Rapamycin‐mediated mouse lifespan extension: Late‐life dosage regimes with sex‐specific effects. ''Aging cell'', ''19''(11), e13269.]</ref> Aged female mice administered rapamycin once every 5 days starting at 20 months of age also extended lifespan.<ref>Arriola Apelo SI, Pumper CP, Baar EL, Cummings NE, '''Lamming DW'''. Intermittent administration of rapamycin extends the lifespan of female C57BL/6J mice. ''J Gerontol A Biol Sci Med Sci''. 2016 Jul; 71(7):876-81. doi: [https://academic.oup.com/biomedgerontology/article/71/7/876/2605199 10.1093/gerona/glw064]. Epub 2016 Apr 18. PMID: 27091134; PMCID: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906329/ PMC4906329]</ref> These studies were important as they suggest that much of the health and longevity benefits of rapamycin could be achieved even when dosed in late life or intermittently, as opposed to only being effective with continual dosing in early life.

=== Yeast ===
Inhibition of TOR signalling by rapamycin significantly increases the lifespan of yeast known as ''Saccharomyces cerevisiae.''<ref>Powers, R. W., Kaeberlein, M., Caldwell, S. D., Kennedy, B. K., & Fields, S. (2006). Extension of chronological life span in yeast by decreased TOR pathway signaling. ''Genes & development'', ''20''(2), 174-184.</ref>

=== Flies ===
Rapamycin extends the lifespan of the fruitfly, ''Drosophila melanogaster.'' The extent of lifespan extension observed is beyond what is achievable by flies undergoing other pro-longevity interventions like dietary restriction, or in mutant flies with mild decrements in insulin/insulin-like growth factor signaling (IIS).<ref>Bjedov, I., Toivonen, J. M., Kerr, F., Slack, C., Jacobson, J., Foley, A., & Partridge, L. (2010). Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. ''Cell metabolism'', ''11''(1), 35-46.</ref> Combining rapamycin with two other drugs that target metabolic pathways, lithium and trametinib, results in additive lifespan extension effects, substantially increasing ''Drosophila'' lifepsan by 48%.<ref>Castillo-Quan, J. I., Tain, L. S., Kinghorn, K. J., Li, L., Grönke, S., Hinze, Y., ... & Partridge, L. (2019). A triple drug combination targeting components of the nutrient-sensing network maximizes longevity. ''Proceedings of the National Academy of Sciences'', ''116''(42), 20817-20819.</ref>

=== Roundworms ===
TOR inhibition by rapamycin extends lifespan in ''Caenorhabditis elegans,'' a roundworm nematode widely used in research areas of the biology of aging. The beneficial effects of rapamycin in ''C. elegans'' seem to be mediated via the SKN-1/Nrf and DAF-16/FoxO pathways.<ref>Robida-Stubbs, S., Glover-Cutter, K., Lamming, D. W., Mizunuma, M., Narasimhan, S. D., Neumann-Haefelin, E., ... & Blackwell, T. K. (2012). TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO. ''Cell metabolism'', ''15''(5), 713-724.</ref>

=== Age-related diseases ===
Rapamycin has been investigated in specific diseases, showing major impacts on reducing mouse cancer risk, cardiac diseases, neurodegenerative-like processes, and many other pathologies.<ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.
</ref>

===== Cancer =====
In the transgenic HER-2/neu mouse model, mice die prematurely due to susceptibility to cancer.<ref name=":6" /> Rapamycin was hypothesized to improve survival in this model due to its ability to slow aging, which would also address an age-related disease like cancer.<ref name=":6" /> The drug was shown to extend maximal lifespan, by delaying aging in multiple different organs and also suppressing cancer development.<ref name=":6">Anisimov, V. N., Zabezhinski, M. A., Popovich, I. G., Piskunova, T. S., Semenchenko, A. V., Tyndyk, M. L., ... & Blagosklonny, M. V. (2010). Rapamycin extends maximal lifespan in cancer-prone mice. ''The American journal of pathology'', ''176''(5), 2092-2097.</ref>

Other studies suggest that rapamycin can extend lifespan in mouse models where cancer naturally develops, such as in mice prone to cancer due to the Apc tumor suppressor gene mutation, or in mice heterozygous for the Rb1 tumor supressor gene, among others.<ref>Hasty, P., Livi, C. B., Dodds, S. G., Jones, D., Strong, R., Javors, M., ... & Sharp, Z. D. (2014). eRapa restores a normal life span in a FAP mouse model. ''Cancer Prevention Research'', ''7''(1), 169-178.</ref><ref>Livi, C. B., Hardman, R. L., Christy, B. A., Dodds, S. G., Jones, D., Williams, C., ... & Sharp, Z. D. (2013). Rapamycin extends life span of Rb1+/− mice by inhibiting neuroendocrine tumors. ''Aging (Albany NY)'', ''5''(2), 100.</ref><ref>Hambright, H. G., Hurez, V., & Curiel, T. J. (2020). Chronic Mechanistic Target of Rapamycin Inhibition: Preventing Cancer to Delay Aging or Vice Versa?. ''Geriatric Oncology'', 111-128.</ref>

A group of investigators in Germany have argued, based on their experiment in C57BL/6J Rj inbred mice, that rapamycin extends lifespan mainly through delaying cancer incidence, instead of via slowed aging.<ref>Neff, F., Flores-Dominguez, D., Ryan, D. P., Horsch, M., Schröder, S., Adler, T., ... & Ehninger, D. (2013). Rapamycin extends murine lifespan but has limited effects on aging. ''The Journal of clinical investigation'', ''123''(8), 3272-3291.</ref> A further analysis of the paper by Johnson et al. pointed to several important limitations of the study, suggesting that such a conclusion may be premature.<ref name=":82">Johnson, S. C., Martin, G. M., Rabinovitch, P. S., & Kaeberlein, M. (2013). Preserving youth: does rapamycin deliver? ''Science translational medicine'', ''5''(211), 211fs40.</ref> Key limitations included the lack of dose-response profiling of rapamycin; studying only the male sex, which is known to respond less to rapamycin likely in part due to sex differences in drug metabolism; lack of reporting on tumor size and incidence, required to determine whether lifespan extension occured only via slowed cancer or from a general effect on aging; and, the cross-sectional nature of the study, which would have reduced sensitivity for detecting age-related organ/tissue changes compared to longitudinal assays.<ref name=":82" /><ref>Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref>Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much? ''Journal of genetics and genomics,'' ''41''(9), 459.</ref>

===== Heart Disease =====
One study investigated the effects of late-life rapamycin dosing in aged female mice, observing a reversal of age-related heart dysfunction.<ref name=":16">Flynn, J. M., O'Leary, M. N., Zambataro, C. A., Academia, E. C., Presley, M. P., Garrett, B. J., ... & Melov, S. (2013). Late‐life rapamycin treatment reverses age‐related heart dysfunction. ''Aging cell'', ''12''(5), 851-862.

</ref> This included benefits to ejection fraction, cardiac hormones, and reduced inflammation, although no effect was observed for heart fibrosis.<ref name=":16" /> Additionally, improvements in behaviour and physical function were demonstrated.<ref name=":16" /> Another study in naturally aged mice showed improvements in cardiac muscle stiffness, diastolic function with rapamycin.<ref name=":7">Quarles, E., Basisty, N., Chiao, Y. A., Merrihew, G., Gu, H., Sweetwyne, M. T., ... & Rabinovitch, P. S. (2020). Rapamycin persistently improves cardiac function in aged, male and female mice, even following cessation of treatment. ''Aging Cell'', ''19''(2), e13086.</ref> Improvements in heart function were shown with only a brief treatment course of 8 weeks.<ref name=":7" /> Benefits persisted even after rapamycin was stopped, which appears consistent with the hypothesis that rapamycin slows aging.<ref name=":7" />

===== Alzheimer's =====
Alzheimer's Disease (AD) is a progressive neurodegenerative disease for which age is the greatest risk factor.<ref>https://www.science.org/doi/10.1126/scitranslmed.aar4289</ref> In an Alzheimer's model of transgenic PDAPP mice, rapamycin was shown to reduce Amyloid-β, one of the hallmarks of AD.<ref name=":8" /> This led to alleviation of AD-like symptoms, such as restored cognition and memory.<ref name=":8">Spilman, P., Podlutskaya, N., Hart, M. J., Debnath, J., Gorostiza, O., Bredesen, D., ... & Galvan, V. (2010). Inhibition of mTOR by rapamycin abolishes cognitive deficits and reduces amyloid-β levels in a mouse model of Alzheimer's disease. ''PloS one'', ''5''(4), e9979.</ref> Similarly, another major AD hallmark known as tau was mitigated by rapamycin in a tauopathy mouse model.<ref name=":14">Ozcelik, S., Fraser, G., Castets, P., Schaeffer, V., Skachokova, Z., Breu, K., ... & Winkler, D. T. (2013). Rapamycin attenuates the progression of tau pathology in P301S tau transgenic mice. ''PloS one'', ''8''(5), e62459.</ref> The mechanism of clearance of these proteins was linked to autophagy, with benefits seen regardless of whether it was dosed early for prevention, or in late life as treatment.<ref name=":14" />

=== Dose response ===
Rapamycin has shown a dose-response in which higher doses produce larger lifespan extension effects. UMHET3 mice of diverse genetic background were treated with varying doses of dietary rapamycin at 4.7, 14, or 42 ppm, revealing that those fed with the highest rapamycin dose had the greatest lifespan extension.<ref>Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref name=":0">[[Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much?. Journal of genetics and genomics= Yi chuan xue bao, 41(9), 459.|Kaeberlein, M. (2014). Rapamycin and aging: when, for how long, and how much? ''Journal of genetics and genomics,'' ''41''(9), 459.]]</ref> Sex differences in response to rapamycin have been hypothesized to also be related to the effective dose, due to male/female differences in drug metabolism. The optimal dose for longevity in mice remains to be seen, but determining this dose will require consideration of the side effect profile of rapamycin.<ref name=":0" />

== Mechanism ==

=== Manipulating metabolic pathways - differences to calorie restriction ===
Rapamycin has often been described as a 'calorie restriction (CR) mimetic'.<ref name=":15">Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., ... & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. ''Aging cell'', ''13''(3), 468-477.</ref><ref name=":4" /> This is in part because CR also inhibits the nutrient-sensing mammalian target of rapamycin (mTOR) pathway.<ref name=":3">Cornu, M., Albert, V., & Hall, M. N. (2013). mTOR in aging, metabolism, and cancer. ''Current opinion in genetics & development'', ''23''(1), 53-62.</ref> mTOR plays key roles in cellular growth in response to amino acids, including effects that inhibit cancer and aging mechanisms.<ref name=":3" /><ref>Johnson, S. C., Rabinovitch, P. S., & Kaeberlein, M. (2013). mTOR is a key modulator of ageing and age-related disease. ''Nature'', ''493''(7432), 338-345.</ref> However, later studies have disentangled the effects of rapamycin from that of caloric restriction, showing that they differ significantly.<ref name=":15" /><ref name=":4">Unnikrishnan, A., Kurup, K., Salmon, A. B., & Richardson, A. (2020). Is rapamycin a dietary restriction mimetic?. ''The Journals of Gerontology: Series A'', ''75''(1), 4-13.</ref> For example, unlike 5 months of CR, rapamycin does not decrease leptin, insulin, IGF-1, or FGF-21 in genetically diverse UM-HET3 mice.<ref name=":15" /> This has important implications for understanding biological aging, including the possibility of using CR and rapalogs in combination therapy to slow aging.<ref name=":4" />

Though distinct from CR, fasting inhibits muscle-specific mTOR signaling with reduced effect in old vs young mice, indicating a poorer autophagy and proteosomal degradation response with age.<ref>White, Z., White, R. B., McMahon, C., Grounds, M. D., & Shavlakadze, T. (2016). High mTORC1 signaling is maintained, while protein degradation pathways are perturbed in old murine skeletal muscles in the fasted state. ''The international journal of biochemistry & cell biology'', ''78'', 10-21.</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/21179166/ Sengupta, S., Peterson, T. R., Laplante, M., Oh, S., & Sabatini, D. M. (2010). mTORC1 controls fasting-induced ketogenesis and its modulation by ageing. ''Nature'', ''468''(7327), 1100-1104.]</ref> However, the ability for rapamycin to inhibit mTOR appears to remain robust throughout life, and significant extension of median and maximal lifespan can be achieved even when treatment is initiated in mid-to-late life.<ref name=":5" /><ref>Blagosklonny, M. V. (2019). Rapamycin for longevity: opinion article. ''Aging (Albany NY)'', ''11''(19), 8048.
</ref> Rapamycin contrasts significantly with CR and fasting, in that the latter could be detrimental when used in late life.<ref>Kemnitz, J. W. (2011). Calorie restriction and aging in nonhuman primates. ''Ilar Journal'', ''52''(1), 66-77.</ref><ref>Goto, S., Takahashi, R., Radak, Z., & Sharma, R. (2007). Beneficial biochemical outcomes of late‐onset dietary restriction in rodents. ''Annals of the New York Academy of Sciences'', ''1100''(1), 431-441.</ref><ref>Kristan, D. M. (2008). Calorie restriction and susceptibility to intact pathogens. ''Age'', ''30''(2), 147-156.</ref> Rapamycin also targets multiple diseases of aging, but seemingly in a segmented, tissue-specific manner.<ref>Neff, F., Flores-Dominguez, D., Ryan, D. P., Horsch, M., Schröder, S., Adler, T., ... & Ehninger, D. (2013). Rapamycin extends murine lifespan but has limited effects on aging. ''The Journal of clinical investigation'', ''123''(8), 3272-3291.</ref>

=== mTORC1 and mTORC2 ===
In non-mammals the mTOR equivalent is known as the target of rapamycin (TOR), first discovered by a team led by Michael Hall in the yeast ''Saccharomyces cerevisiae''.<ref>Thomas, G., & Hall, M. N. (1997). TOR signalling and control of cell growth. ''Current opinion in cell biology'', ''9''(6), 782-787.</ref><ref>Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., & Hall, M. N. (1993). Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. ''Cell'', ''73''(3), 585-596.</ref> mTOR signalling appears to be evolutionarily conserved, and this extends further to include various mammals, such as mice, rats, and dogs.

Rapamycin acts on mTOR, with multiple signaling functions subdivided across two major protein complexes known as mTORC1 and mTORC2.<ref name=":3" /> There is some evidence suggesting that the health and lifespan benefit of rapamycin is more related to inhibition of mTORC1 than mTORC2.<ref name=":19">Lamming, D. W., Ye, L., Katajisto, P., Goncalves, M. D., Saitoh, M., Stevens, D. M., ... & Baur, J. A. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. ''science'', ''335''(6076), 1638-1643.</ref><ref name=":11">Lamming, D. W., Mihaylova, M. M., Katajisto, P., Baar, E. L., Yilmaz, O. H., Hutchins, A., ... & Sabatini, D. M. (2014). Depletion of Rictor, an essential protein component of m TORC 2, decreases male lifespan. ''Aging cell'', ''13''(5), 911-917.</ref> In mice, males exhibit weaker lifespan extension effects from rapamycin than in females. One study suggests that inhibiting mTORC2 explains why the sex difference in the response to mTOR inhibition by rapamycin.<ref name=":11" />

=== Effects on glucoregulatory control ===
A noted issue regarding rapamycin is the disruption of glucose metabolism with chronic dosing, which has been observed in both humans and mice. In mice, this side effect has been shown to be due in part to disruption of mTORC2 in the liver, leading to hepatic insulin resistance.<ref name=":19" /> This effect has previously been shown to be reversible upon stopping the drug in both lean and obese mice.<ref name=":18">Liu, Y., Diaz, V., Fernandez, E., Strong, R., Ye, L., Baur, J. A., ... & Salmon, A. B. (2014). Rapamycin-induced metabolic defects are reversible in both lean and obese mice. ''Aging (Albany NY)'', ''6''(9), 742.</ref> Whether disrupted glucose metabolism is dispensable for the lifespan extending effects of rapamycin remains controversial.<ref>Blagosklonny, M. V. (2012). Once again on rapamycin-induced insulin resistance and longevity: despite of or owing to. ''Aging (Albany NY)'', ''4''(5), 350.</ref><ref>[https://www.nature.com/articles/s41419-019-1822-8 Blagosklonny, M. V. (2019). Fasting and rapamycin: diabetes versus benevolent glucose intolerance. ''Cell death & disease'', ''10''(8), 1-10.]</ref><ref>[https://www.science.org/doi/full/10.1126/science.1215135 Lamming, D. W., Ye, L., Katajisto, P., Goncalves, M. D., Saitoh, M., Stevens, D. M., ... & Baur, J. A. (2012). Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity. ''science'', ''335''(6076), 1638-1643.]</ref><ref>Strong, R., Miller, R. A., Antebi, A., Astle, C. M., Bogue, M., Denzel, M. S., ... & Harrison, D. E. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α‐glucosidase inhibitor or a Nrf2‐inducer. ''Aging cell'', ''15''(5), 872-884.</ref> Rapamycin has previously been shown to increase insulin sensitivity with acute dosing, while decreasing insulin sensitivity with chronic dosing.<ref>Ye, L., Varamini, B., Lamming, D. W., Sabatini, D. M., & Baur, J. A. (2012). Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2. ''Frontiers in genetics'', ''3'', 177.</ref>

=== Reducing the effects of cellular senescence ===
The accumulation of senescent cells is thought to be an important mechanism underlying aging. Rapamycin is regarded as a senomorphic that may inhibit the pro-inflammatory secretory phenotype produced by senescent cells in humans, mice, and rats.<ref>[[Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., ... & Perez, V. I. (2017). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism. Aging cell, 16(3), 564-574.|Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., ... & Perez, V. I. (2017). Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism. ''Aging cell'', ''16''(3), 564-574.]]</ref><ref>Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.
</ref> A preliminary study in humans aged 40 years or older showed that topical rapamycin reduced markers of cellular senescence in the skin and improved its physical appearance.<ref>[https://doi.org/10.1007%2Fs11357-019-00113-y Chung, C. L., Lawrence, I., Hoffman, M., Elgindi, D., Nadhan, K., Potnis, M., ... & Sell, C. (2019). Topical rapamycin reduces markers of senescence and aging in human skin: an exploratory, prospective, randomized trial. ''Geroscience'', ''41''(6), 861-869.]
</ref>

== Human clinical trials ==
[[File:Rapamycin.jpg|thumb|235x235px|Part of the rationale of the PEARL study is to determine the optimal dose of rapamycin to potentially slow aging.<ref name=":2" />]]

=== PEARL study ===
Rapamycin is currently being tested for safety and efficacy in a clinical trial called the Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity (PEARL) study. The clinical trial aims to systematically investigate the use of rapamycin to promote healthy longevity, and is expected to conclude in 2023.<ref>[https://clinicaltrials.gov/ct2/show/NCT04488601 ''Participatory Evaluation (of) Aging (With) Rapamycin (for) Longevity Study - Full Text View - ClinicalTrials.gov''. Clinicaltrials.gov. (2021). Retrieved 27 May 2021, from https://clinicaltrials.gov/ct2/show/NCT04488601.]</ref>

The study will begin with 200 adults aged 50 years or older who will receive rapamycin for up to one year. The study is being conducted by AgelessRx, a new company dedicated to developing scientifically supported interventions to prevent and treat age-related diseases, in collaboration with the University of California.<ref>[https://www.longevity.technology/agelessrx-and-funding-an-irb-approved-rapamycin-trial/ ''AgelessRx and funding an IRB-approved rapamycin trial - Longevity.Technology''. Longevity.Technology. (2021). Retrieved 27 May 2021, from https://www.longevity.technology/agelessrx-and-funding-an-irb-approved-rapamycin-trial/.]</ref>

The trial aims to obtain clinical data at 6 and 12 months of treatment, such as via testing of blood, body composition DXA, fecal microbiome, immune function, inflammation, skeletal muscle, and epigenetic aging clocks.

== Dog clinical trials ==
The Dog Aging Project is a US Government NIH-funded initiative investigating dog aging.<ref>https://dogagingproject.org/</ref> The project is led by Professor Matt Kaeberlein at the University of Washington.<ref name=":10">Kaeberlein, M., Creevy, K. E., & Promislow, D. E. (2016). The dog aging project: translational geroscience in companion animals. ''Mammalian genome'', ''27''(7), 279-288.</ref>

The Test of Rapamycin In Aging Dogs (TRIAD) study is investigating rapamycin as a treatment to slow aging in dogs. The investigators hope to increase healthy canine lifespan with rapamycin by delaying the onset of age-related diseases like cancer and heart disease.<ref name=":9" /><ref name=":10" />

Aging biology scientists believe that studying dog aging might not only help improve canine healthspan, but also have implications for humans.<ref name=":10" /> Dogs may be a useful animal model because they share the same environment that humans live in, and suffer from similar chronic diseases with aging.<ref>Hoffman, J. M., Creevy, K. E., Franks, A., O'Neill, D. G., & Promislow, D. E. (2018). The companion dog as a model for human aging and mortality. ''Aging Cell'', ''17''(3), e12737.</ref>

===== Heart disease =====
One randomized-controlled trial in 24 middle-aged dogs treated with low-dose rapamycin showed suggestion of partial reversal of age-related heart dysfunction, as measured via echocardiography. The intervention was well-tolerated, with no clinically meaningful adverse events noted with a non-immunosuppressive dose of rapamycin during the 10 week period. This was a small study over a relatively short duration; further testing in larger clinical studies will be necessary to determine whether rapamycin can be used to treat age-related heart disease in dogs.

== Regulatory approval ==
Rapamycin was approved by the US Food and Drug Administration (FDA) in 1999 to prevent organ rejection in liver transplant patients, and has been marketed under the brand name Rapamune.<ref>[https://link.springer.com/article/10.1007/s11357-020-00274-1 Selvarani, R., Mohammed, S., & Richardson, A. (2020). Effect of rapamycin on aging and age-related diseases—past and future. ''GeroScience'', 1-24.]</ref> The patent on rapamycin has expired, and chemically similar compounds called 'rapalogs' are being researched by biotechnology companies.<ref>[https://pubs.acs.org/doi/full/10.1021/acsmedchemlett.9b00215 Abdel-Magid, A. F. (2019). Rapalogs potential as practical alternatives to rapamycin.]
</ref> It is not currently approved for use as an anti-aging medication, due to lack of human clinical data for this purpose.

== Safety ==
Rapamycin has been used to treat millions of patients over several decades since obtaining FDA approval in 1999. It is generally considered safe in humans, but only when used under clinical supervision for specific indications. Various side effects have been reported with the high dose of rapamycin used to prevent rejection in organ transplant patients, who are often concurrently treated with multiple other medications.<ref>Webster, A. C., Lee, V. W., Chapman, J. R., & Craig, J. C. (2006). Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials. ''Transplantation'', ''81''(9), 1234-1248.</ref> These include pain, headache, fever, high blood pressure, glucose intolerance, nausea, abdominal pain, constipation, diarrhea, thrombocytopenia, leukopenia, among others. However, side effects are mostly reversible and represent worst-case scenarios, particularly because the patients sampled in clinical studies are already severely ill and taking high doses of the drug along with other medications.<ref>Bischof, E., Siow, R.C., Zhavoronkov, A. and Kaeberlein, M., 2021. The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19. ''The Lancet Healthy longevity'', ''2''(2), pp.e105-e111.
</ref>

Case studies of the safety profile of rapamycin in the context of overdosing have suggested that it may have a large margin of safety or a high median lethal dose, but only in the acute setting.<ref>Ceschi, A., Heistermann, E., Gros, S., Reichert, C., Kupferschmidt, H., Banner, N. R., ... & Taegtmeyer, A. B. (2015). Acute sirolimus overdose: a multicenter case series. ''PLoS One'', ''10''(5), e0128033.</ref> The distinction with chronic high dose mTOR inhibition must be made because resultant immunosuppression can lead to susceptibility to infection with fatal consequences. Some preclinical data suggests that the longevity benefits of rapamycin may be retained via intermittent dosing.<ref name=":2" /> In considering the known clinical data about rapamycin's controversial safety at continuous, high doses, some researchers have proposed that rapamycin should dosed intermittently to minimize side effects while sufficiently inhibiting mTOR for an effect on aging.<ref name=":2" />

From a longevity perspective, there is a lack of published clinical data demonstrating the safety of rapamycin in healthy adults. One randomized pilot study of rapamycin in 25 older adults aged 70-95 showed no clinically significant effects, including a lack of effect on immune function.<ref name=":13">Kraig, E., Linehan, L. A., Liang, H., Romo, T. Q., Liu, Q., Wu, Y., ... & Kellogg Jr, D. L. (2018). A randomized control trial to establish the feasibility and safety of rapamycin treatment in an older human cohort: Immunological, physical performance, and cognitive effects. ''Experimental gerontology'', ''105'', 53-69.</ref> This was a small study with a low dose of rapamycin, dosed over a short duration of 8 weeks. Considering the fact that, based on preclinical animal data, any potential benefit of rapamycin for aging will require long-term dosing, further testing in clinical trials is necessary to better characterize safety.<ref name=":13" />

Clinical trials such as the PEARL study are needed to provide evidence for the safety profile of rapamycin in otherwise healthy older adults.

== Rapalogs ==
Rapalogs are molecules with similar mechanism to rapamycin, primarily via mTORC1 inhibition. These drugs are generally predicted to function similarly to rapamycin in enhancing lifespan and reducing age-related decline in physiological function. However, only one rapalog, everolimus, has published clinical data in this context.<ref>[https://www.thelancet.com/servlet/linkout?suffix=e_1_5_1_2_55_2&dbid=8&doi=10.1016/S2666-7568(20)30068-4&key=24379984&cf= Kaeberlein, M. (2013). mTOR inhibition: from aging to autism and beyond. ''Scientifica'', ''2013''.]</ref> RTB101 has also been described as a selective mTOR inhibitor, but some controversy exists.<ref>Kaeberlein, M. (2020). RTB101 and immune function in the elderly: interpreting an unsuccessful clinical trial. ''Translational Medicine of Aging'', ''4'', 32-34.</ref><ref>Bischof, E., Siow, R. C., Zhavoronkov, A., & Kaeberlein, M. (2021). The potential of rapalogs to enhance resilience against SARS-CoV-2 infection and reduce the severity of COVID-19. ''The Lancet Healthy longevity'', ''2''(2), e105-e111.</ref>

=== mTOR inhibition improves immune function in the elderly ===

In a phase 2 randomized clinical trial published in ''Science Translational Medicine'' in 2014, low-dose TORC1 inhibition with the rapalog everolimus showed improvement in immune function in the elderly. The clinical trial enrolled 218 adults aged ≥65 years, observing decreased incidence of all infections, as well as improved influenza vaccination responses and upregulation of antiviral immunity.<ref name=":17">Mannick, J. B., Del Giudice, G., Lattanzi, M., Valiante, N. M., Praestgaard, J., Huang, B., ... & Klickstein, L. B. (2014). mTOR inhibition improves immune function in the elderly. ''Science translational medicine'', ''6''(268), 268ra179-268ra179.</ref>

Everolimus enhanced the influenza vaccine response by approximately 20% at relatively well tolerated doses.<ref name=":17" /> One mechanism was related to a reduction in the percentage of CD4 and CD8 T cells expressing the programmed death-1 receptor, which has increased expression with age and a major role in inhibiting T cell signaling.<ref name=":17" /> These findings suggest that, at an appopriate dose, mTOR inhibition may improve the age-related decline in immune function in the elderly.

=== TORC1 inhibition enhances immune function and reduces infections in the elderly ===

A phase 2a trial clinical trial randomized 264 older adults to treatment with everolimus and placebo, and was published in ''Science Translational Medicine'' in 2018. The trial showed potential for reducing the effects of immune aging, with improvement in influenza vaccination response in the elderly.<ref name=":1">Mannick, J. B., Morris, M., Hockey, H. U. P., Roma, G., Beibel, M., Kulmatycki, K., ... & Klickstein, L. B. (2018). TORC1 inhibition enhances immune function and reduces infections in the elderly. ''Science translational medicine'', ''10''(449).</ref>[[File:Rtb101 Ph2 Ph3.jpg|thumb|461x461px|A) Number of patients with laboratory-confirmed RTIs of ''any severity'' caused by specific viruses, comparing RTB101 group (blue) versus placebo (grey) in the phase 2b, phase 3, and combined phase 2b/3 RCTs B) Number of patients with laboratory-confirmed RTIs with ''severe symptoms'' caused by specific viruses in the RTB101 group (blue) versus placebo (grey) in the phase 2b, phase 3, and combined phase 2b/3 trials. RTI = respiratory tract infection]]

=== Improving immune function in older adults for respiratory tract infections, including coronaviruses ===
Low-dose mTOR inhibition with dactolisib in a Phase 2b and phase 3 trial in the elderly showed reduced coronavirus (non [[COVID-19]]) incidence, as well as reductions in severe symptoms.<ref name=":12">Mannick, J. B., Teo, G., Bernardo, P., Quinn, D., Russell, K., Klickstein, L., ... & Shergill, S. (2021). Targeting the biology of ageing with mTOR inhibitors to improve immune function in older adults: phase 2b and phase 3 randomised trials. ''The Lancet Healthy Longevity'', ''2''(5), e250-e262.</ref> However, the data remains inconclusive as the study was powered statistically for a reduction in clinically symptomatic respiratory tract infections (RTIs), and not laboratory-confirmed RTIs.<ref name=":12" />

Following the success of two phase 2 clinical trials investigating mTOR inhibition for targeting the aging immune system, dactolisib is currently being pursued for the treatment of COVID-19 in a phase 2a placebo-controlled trial (ClinicalTrials.gov Identifier: NCT04584710, NCT04409327), exploring the potential for preventing severe disease in elderly adults with no symptoms, who have been exposed to [[COVID-19]].<ref name=":12" />

Unpublished data from the phase 2 trial of RTB101 for COVID-19 among nursing home patients treated within 3 days from testing positive saw promising results. None of those treated with RTB101 developed symptoms (n=18), while the placebo treated control group had 4 severe cases of disease and 2 deaths. While this was a statistically significant finding, larger trials are warranted for further evidence of potential benefit.

This trial is being run by the biopharmaceutical company resTORbio and has obtained funding from the National Institute on Aging (NIA/NIH). The studies with dactolisib for COVID-19 is one of several clinical trials in the aging biology field aiming to target aging to improve the aging immune system.<ref name=":12" />

== References ==
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[[Category:Pillar Articles]]
[[Category:Drugs]]