New Results From A Rigorous Life Extending Drug Testing Program

The National Institute of Aging (NIA) Interventions Testing Program (ITP) is the most rigorous program for testing the effects of drugs on the lifespan of mice. What makes this project unique is that all interventions are tested in parallel in three different sites: the Jackson Laboratory, University of Michigan, and the University of Texas Health Science Center at San Antonio using identical protocols. Doing the same mice study in parallel in three different labs and then comparing the results is virtually unheard of in biomedical research given the cost of doing the study in threefold. 

What do we know already?

So far, in the 11 years that this project is running, they have conducted 53 lifespan tests with 30 different compounds. Most readers are probably familiar with the fact that the drug rapamycin robustly extends lifespan in mice. Rapamycin was one of the unexpected early outcomes of the ITP project. The researchers had problems with formulating a diet that contained rapamycin. By the time that they had discovered the solution (microencapsulation) the mice destined to be used for this experiment had already reached middle age. The researchers decided to go ahead anyway and give the drug to these old mice expecting to see no effect.

Most people at the time believed that life extending drugs would only work when initiated early in life. To their large surprise the rapamycin treated mice lived about 13% longer. The ITP has since repeated the rapamycin experiment multiple times, including starting at young age and using different concentrations, and every time this drug extended lifespan. So far the ITP has found significant lifespan extension in at least one sex for the following 6 compounds: aspirin, NDGA, rapamycin, acarbose, methylene blue, and 17-α-estradiol.

A new round of testing

This time the following compounds were tested: 

1. 17-α-estradiol: Is a form of the female hormone estrogen that has been reported to have neuroprotective properties (e.g. stroke model, Parkinson’s disease,…). It was previously tested and only extended lifespan in male mice but with a large variation between the three labs. The researchers speculated that the drug might have failed to extend the lifespan of female mice because the dose was too low. Therefore they decided to test it again at a higher concentration (now 14.4 ppm versus 4.8 ppm before). 
2. Protandim ®: Is a mixture of 5 botanical extracts (milk thistle, bacopa, ashwagandha, green tea, and turmeric). This mixture was designed to be an excellent activator of a signaling pathway known as Nrf2/ARE involved in upregulation of antioxidant and anti-electrophilic stress defenses. 
3. Metformin: Is a drug used for the treatment of type 2 diabetes mellitus. Multiple previous reports show that it extends lifespan and a human trial is under way (read our Longevity Reporter article on this trial here.
4. Rapamycin: This drug is used for preventing the rejection of transplanted organs and is currently one of the most proven life extension drugs with at least 8 studies showing that it extends lifespan in rodents. The goal in this study was to see if the combination of rapamycin with metformin gave any benefit over rapamycin alone. Read more abour rapamycin here.   
5. Ursodeoxycholic acid (UDCA): Is a bile acid that had been shown to prevent the recurrence of colon cancer and prevented experimentally-induced diabetes in rats. Bile acids are compounds that our body secretes in the intestinal tract to help with the absorption of dietary lipids. 
6. Nordihydroguaiaretic acid (NDGA): Is a compound extracted from the creosote bush. It’s a powerful antioxidant that has previously been shown to extend lifespan but only in male mice. In this study a higher (5000 ppm) and a lower dose (800 ppm) than previous time (2500 ppm) was tested. 
7. Fish oil: Everyone knows fish oil. It’s supposed to be good for your heart but is it also good for your lifespan? 
8. Acarbose: This prevents you from breaking down complex sugars into simple sugars that can be absorbed in your digestive tract and hence lowers blood sugar levels. It’s used for the treatment of type 2 diabetes mellitus. Acarbose was previously shown to extend the lifespan of UM-HET3 mice when started at 4 months of age. In this study the researchers wanted to test if it also extends lifespan when started at 16 months of age. 

The creosote bush, source of nordihydroguaiaretic acid.

17-α-estradiol significantly extended the mean and maximum lifespan (oldest 10%) in male but not female mice. Protandim significantly extended the mean lifespan of male mice and also extended the maximum lifespan of male mice but this did not reach significance. It also extended the mean and maximum lifespan of female mice but neither reached significance. Metformin increased mean lifespan in male mice but this did not reach significance and had no effect on maximum lifespan in males and mean or maximum lifespan in females. UDCA followed the same pattern as metformin. Acarbose significantly extended the mean and maximal lifespan of male mice and the maximum lifespan of female mice. The mean lifespan of female mice was also extended but this did not reach statistical significance. 

It was surprising to see that metformin did not improve lifespan despite that this study used the same concentration (0.1%) as a previous study published in Nature Communications that found that metformin at this dose lead to a significant increase of lifespan in male C57BL/6 mice (+5.8%) and a non-significant increase of 4.15% in male B6C3F1 mice. Although it should be noted that the current study used a different statistical tool to calculate significance than in the Nature Communications paper. In that study the statistical tool used gave more weight to deaths at earlier ages than in the current paper. In conclusion based on the data discussed above, only 17-α-estradiol (but only in males) and acarbose had significant effects on lifespan.   

The effect of multiple interventions in the lifespan of male and female UM-HET3 mice. The stars above the graph shows that statistical significance (defined as p < 0.05) was reached. Image credit: Sven Bulterijs. Based on data from Strong et al. (2016)

NDGA significantly extended the mean lifespan of male mice at all three concentrations (800 ppm, 2500 ppm, and 5000 ppm). However it did not extend maximum lifespan in male mice and slightly decreased the mean and maximum lifespan of female mice (although this was not significant). Stephen Spindler also tested NDGA in male mice (female mice were not tested) and similarly found an increase in mean but not maximal lifespan. The researchers also tested the effect of NDGA on grip duration and on the time that mice can stay on a rotating rod (the rotarod test). Grip duration declines with age but in NDGA-treated mice the grip duration of elderly male mice was equal to that of young mice. Grip duration was significantly improved by NDGA treatment. NDGA treatment also extended the period that both male and female mice stayed on the rod in the rotarod test.

A Rotarod test machine. The rod rotates and the time that mice can stay running on it is measured. This is a measure of the mice’s balance, coordination, physical condition, and motor-planning

Fish oil did not reach significance in any test but at 15,000 ppm it did have a non-significant positive effect on lifespan in male mice. In contrast it decreased mean and maximum lifespan in female mice at 15,000 ppm and in male mice at 50,000 ppm although none reached significance. When looking at the data for each lab individually, it was found that fish oil at 50,000 ppm lead to a significantly decreased lifespan (by 18%) at the University of Michigan. A previous study by Stephen Spindler had already discovered that treatment of long-lived mice with a medical-grade fish oil and krill oil shortened their lifespan although it did not reach significance. These data fall in line with the prediction from the membrane hypothesis that says that a higher concentration of easily oxidizable polyunsaturated fats in the cell membrane should have a negative effect on lifespan by increasing the production of damaging lipid peroxides and lipid-derived electrophiles. 

The effect of NDGA and fish oil in different concentrations on the lifespan of male and female UM-HET3 mice. The stars above the graph shows that statistical significance (defined as p < 0.05) was reached. Image credit: Sven Bulterijs. Based on data from Strong et al. (2016)

As discussed above metformin alone had no effect on lifespan. But when metformin was combined with rapamycin the mean and maximum lifespan was significantly extended both in male and female mice. The researchers did not set up a separate rapamycin trial this time so they compare the data versus their previous trials (in which rapamycin was started either at 9 months of age or at 20 months of age) with rapamycin that used the same mouse model and same dose of rapamycin (years 2006 and 2009). When taking these data together and doing statistical analysis the addition of metformin to the rapamycin did not lead to a significant effect over rapamycin alone. The rationale for combining metformin with rapamycin is the fact that chronic rapamycin treatment has been shown to cause glucose-intolerance (diabetic-like changes) and cause hyperlipidemia (increase in blood lipids) both of which are improved by metformin.  

The effect of metformin and rapamycin either separately or combined on the lifespan of male and female UM-HET3 mice. The stars above the graph shows that statistical significance compared to control (defined as p < 0.05) was reached. Image credit: Sven Bulterijs. Based on data from Strong et al. (2016), Harrison et al. (2009), and Miller et al. (2011)

In conclusion this new study has confirmed that 17-α-estradiol, nordihydroguaiaretic acid, and acarbose extends lifespan in UM-HET3 mice. In addition this study provided for the first time evidence that the botanical mixture protandim extends lifespan but this only reached significance for mean lifespan in male mice. While metformin alone failed to increase the lifespan of mice, the combination of metformin and rapamycin lead to a significant increase in mean and maximum lifespan for male and female mice. The combination of rapamycin and metformin had a bigger effect on lifespan than just rapamycin alone but the difference did not reach significance. However this was based on comparing old data (rapamycin alone) with new data (rapamycin + metformin) and hence we should be careful to draw conclusions on this. 

References:

Harrison DE et al. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460: 392-395. 

Miller RA et al. (2011). Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogenous mice. J Gerontol A Biol Sci Med Sci 66A: 191-201. 

Martin-Montalvo A et al. (2013). Metformin improves healthspan and lifespan in mice. Nature Communications 4: 2192. 

Harrison DE et al. (2014). Acarbose, 17-α-estradiol, and nordihydroguaiaretic acid extend mouse lifespan preferentially in males. Aging Cell 13: 273-282. 

Spindler SR, Mote PL, Flegal JM (2014). Dietary supplementation with Lovaza and krill oil shortens the life span of long-lived F1 mice. Age (Dordr.) 36(3): 9659. 

Spindler SR et al. (2015). Nordihydroguaiaretic acid extends the lifespan of drosophila and mice, increases mortality-related tumors and hemorrhagic diathesis, and alters energy homeostasis in mice. J Gerontol A Biol Sci Med Sci 70: 1479-1489. 

Strong R et al. (2016). Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer. Aging Cell [Epub ahead of print].