Discussion

Although current research foreshadows a promising perspective for using metformin and rapamycin as anti-aging drugs, there are still some concerns that need to be highlighted, and they apply not only to the researches of metformin and rapamycin, but to other anti-aging mechanism and anti-aging drug researches as well.
First, despite the positive outcomes from many studies, it is not uncommon to find a change in dosage turning the result from life-extending to life-ending. When a low dose of metformin (0.1%) was given to middle-aged male mice with their diet, their lifespans were extended by 5.83% on average, but a higher concentration (1%) became toxic.21 In another study, although metformin activated AMPK and suppressed lipid storage in fruit flies, their lifespan did not increase. At higher doses (25mM and 50mM), metformin reduced the survival rates. The authors reasoned the causes to be excessive starvation, disrupted intestinal fluid homeostasis, or metformin toxicity.97 In the PD study with mice models created with MPTP, when MPTP and metformin were given in the same day, 75% lethality ensued in the mice. Furthermore, although metformin increased the levels of BDNF and GDNF, two neurotrophic factors, the high dosage (400 mg/kg) killed all the mice.95 The issues with dosage along with physical and genetic differences between humans and animals make scaling the positive lab results for human use a tricky matter. In the study that showed metformin’s beneficial effects for treating CVDs in mice, the dosage was 200mg/kg, a number that can no way be applicable to humans.41 Hence, conducting human clinical trials may be a more efficient approach to find a safe and effective dosage for human use. Encouragingly, when anti-diabetic doses of metformin were given to 12 pre-operative endothelial cancer patients and comparison of their tissue samples before and after the operation were made, the same effects observed in vitro were found – increased AMPK phosphorylation, decreased tumor cell proliferation, and decreased H19 levels.19
Another issue that stands in the way is the side effects associated with chronic use of drugs. About 25% of patients treated with metformin have gastrointestinal side effects associated with the phenotype of organic cation transporter 1 (OCT1).98 Besides, chronic use of metformin can cause dose-dependent vitamin B12 deficiency, increasing the risk for anemia and neuropathy.99,100 Lactic acidosis has been reported as a side effect of metformin, but there has been controversies, and in the study using diabetes model mice to study AD-like brain changes, metformin did not further increase the serum lactate concentrations.81 Whether this holds true in healthy mice or humans is yet to be seen. As for rapamycin, over a third of users have reported diarrhea and nausea, accounting for around 5% of treatment discontinuation.101 The issues with side effects can be addressed in four ways: the first is to selectively take supplements, such as vitamin B12, to make up for the loss. The second is to reduce the dose and increase the interval between every dose. A small RCT suggests short-term use of rapamycin to be relatively safe approach.102 Intermittently administering 2mg/kg of rapamycin every 5 days has also reduced incidence of side effects in mice and extended their lifespan.103 More clinical trials are needed to calibrate the balance between safety and anti-aging effectiveness. The third way is taking a variety of anti-aging drugs (also known as drug cocktail therapy), each with a very low dose, instead of taking only metformin or rapamycin since the side effects are dose-dependent. Although cultured cells and a mice study both showed metformin combined with insulin reduced A-β peptide levels,80,81 the GPRD study showed long-time combined exposure to metformin and other anti-diabetic drugs increased the risk of AD while using only metformin did not show any difference.104 Therefore, this method requires further validation as well. The fourth way is to find analogs with fewer side effects. Recent study found DL001, an effective mTORC1 specific rapalog, does not induce metabolic disruption and immunesuppression.105 Furthermore, a low dose of rapalog (RAD001) combined with the catalyst BEZ235 reduced infection by 40% and improved response to influenza vaccination in healthy elderly (aged 65 and older) subjects. More importantly, this combination therapy was well-tolerated in majority of the subjects.106Rapalogs have been approved for treating multiple cancers, including renal cell carcinoma, hepatocellular carcinoma and mantle cell lymphoma, making it by far the most promising way to circumvent the side effects of rapamycin.107,108 Nevertheless, as reported clinical benefits have been modest,58,59 the quest for more effective rapalogs is still ongoing. On the other hand, metformin had fewer available analogs with well-studied side effects. Phenformin and buformin, which are biguanide drugs like metformin, were withdrawn from the market due to fatal lactic acidosis.109Mito-metformin, synthesized by adding a positively charged triphenylphosphonium group to metformin, showed 100-fold to 1000-fold more anti-proliferative effects depending on alkyl chain lengths, but how the drastically improved potency will impact healthy cells is poorly understood at the moment.110
Future research should also work to elucidate how gender influences drug effectiveness. Metformin increased mean lifespan of female mice by 4.4% while decreased that of male mice by 13.4%.111 Male pre-diabetic patients who received metformin had a significantly lower coronary calcium score compared with control while the female group did not.46 Such sexual dimorphism also affects the lifespan of mice that took rapamycin – female mice had greater lifespan increase than male mice did.24,112 These studies have showed varied amount by which gender influences drug effectiveness, and studying drug-hormone interactions could help finding the reason.
Besides these issues, much more can be found about the genetic mechanisms that regulate lifespan. Although many positive outcomes have come out of attempts to control DNA methylation with metformin and rapamycin, the full picture of epigenetic modifications have not been understood. Metformin treatment led to a combination of hyper, hypo, and non-differentially methylated CpG sites, and this was due to a combination of direct and indirect effects. For example, hypermethylation of one site can lead to reduced expression of a protein, and this can have downstream effects that alter methylation status of other sites.19 Understanding this complex network of interactions will not only promote further understanding of metformin and rapamycin, but also help developing more anti-aging measures. APOE, a locus on chromosome 5q33.3, and FOXO3A are all known to correlate with longevity. It has also been mentioned above that a GWAS identified 5 SNPs related to cerebellum aging.83In addition, SNPs in the human genome also affects the efficacy of drugs. For example, rs2740574, located in CYP3A4 changes breast cancer cells’ response to rapamycin by altering drug metabolisms in liver, and rs2282143, located in SLC22A1, changes breast cancer cells’ response to metformin by affecting the rate of drug entering cells.113 A locus on chromosome 11 (rs11212617) is associated with the glycemic response to metformin.114Three SNPs (rs8111699, rs11212617, rs9803799), which are located in the LKB1, ATM and PRKAA2, have been identified as significant influencers on metformin therapy by affecting the AMPK pathway.115–117 As DNA sequencing becomes more convenient and accessible, it is reasonable to assume that increasing amount of genetic data and research efforts will reveal many more such connections, and they can point to novel genes and drug targets or be used for precision medicine to improve current treatments.
The issues of dosage, side effects, sexual dimorphism, and genetic regulatory mechanisms all point to the need for a large-scale clinical trial. The Targeting Aging with Metformin (TAME) trial is a large placebo-controlled trial that has been designed to enroll 3000 subjects to test whether metformin delays age-related diseases.118 The TAME trial received FDA approval in 2015, and after receiving all the required budget in 2019, it was set to start at the end of the same year. The TAME trial may make metformin the first approved drug for anti-aging, but more importantly, since it is not testing metformin against a single disease but a collection of age-related ones, it establishes aging as a medical condition that can be intervened or treated instead of an irreversible process outside human control. The shift in the notion of aging will make future anti-aging clinical trials proceed with much more ease.119