Introduction

Aging (or senescence) has been the topic of many scientific research in recent years. Reaching an advanced age while remaining in good health was for a long time considered as a utopia. Nowadays, scientists are trying to analyze, understand and slow down the causes of aging. 

The fight against aging is also a fight with our metabolism and the different causes that accelerate this process. In this text, we will explore the nine main causes of aging in humans, as described in a well-known article by scientists: Hallmarks of aging published in the journal Cell in 2013.

These nine causes have been subdivided into three subclasses: the primary causes, the antagonists of aging, and finally the secondary causes of aging that result from the first two.

 Primary causes

1. DNA damage

DNA is the carrier of the genetic material that confers certain specific characteristics to the human being. It is made up of a large number of nucleotides that serve to encode the genetic information of living beings into gene  forms. The genome is the unique combination of a person’s genes, or genetic characteristics. It is located primarily in the nucleus of eukaryotic cells.

During cell division (mitosis), the genetic information contained in the mother cell will duplicate and be transmitted to each of the daughter cells, a process defined as DNA replication.

Certain life habits (cigarettes, food…) and other internal or external causes can affect the proper functioning of the genome over time, and thus accumulate errors in DNA replication. These errors can either repair themselves, or cause the death of the mother cell (apoptosis  ) or be transmitted to the daughter cells.

Repair of DNA damage:

Our body contains certain genes that make proteins to repair the damage done during DNA replication. But the question is, “what happens to this repair system when our body reaches a certain age?”

The various coenzymes that participate in the repair mechanism of genome damage tend to decrease with age. Among them, NAD+.  Several studies have been done in this field, with the aim of finding a correlation between our NAD+ level and the capacity of our organism to repair lesions in the DNA.

What is important to remember is that lesions in the DNA during replication can either lead to cell death or replicate defective cells. The accumulation of these lesions is one of the primary causes of aging.

2. Telomere shrinkage

Telomeres are regions of DNA called “non-coding” (sequences of the genome that do not produce proteins), located at the end of each chromosome and whose main function is to protect the chromosomes.

During cell division, the DNA polymerase enzyme, responsible for DNA duplication, does not code the last nucleotides at the ends of the chromosomes, thus the telomeres. Thus, with each division, the telomeres shorten a little more until they disappear completely. With the disappearance of telomeres, the cell stops its division and dies. Some of our cells such as stem cells, germ cells and some of our somatic cells express an enzyme called telomerase. This enzyme is able to maintain the length of telomeres during cell division by adding a specific sequence of nucleotides to the ends of the chromosomes. Because of this mechanism, cells containing telomerase can replicate indefinitely.

Unfortunately, 90% of cancer cells express telomerase, which makes them capable of replicating forever as well.

The shrinking of telomeres causes our cells to die and our tissues to age. This implies an important place in anti-aging research.

3. Epigenetics

Epigenetics is the set of mechanisms that can modify the expression of genes without changing their DNA sequence.

Three main epigenetic modes of action that can influence lifespan have been identified:

– DNA methylation: a phenomenon that consists of adding or removing a methyl molecule in certain DNA sequences. The expression of genes can be modified by these changes which, by accumulating, can cause certain problems such as an increase in cholesterol levels or an increase in the risk of cardiovascular diseases.

– Histone acetylation: Histone acetylation and deacetylation are essential components of gene regulation.  Acetylation, like methylation, is the process by which an acetyl functional group is transferred from one molecule to another. Deacetylation is the opposite, an acetyl group is removed from a molecule. These changes, too, can have some effect on our bodies and alter gene expression.

– Chromatin remodeling: Chromatin is the structure within which DNA is packaged. Chromatin can be remodeled, condensed. In its most condensed form it is called heterochromatin, in its least condensed form it is called euchromatin. An imbalance between heterochromatin and euchromatin can occur with age. This imbalance will change not only the stability of the chromosomes, but also the expression of the genes.

These epigenetic alterations can therefore modify gene expression and cause certain age-related diseases.

4. Protein folding

Proteins are present in every gene in our genome. The folding of proteins on themselves allows them to perform their multiple functions in our body. When protein folding is not done properly, it can be due to either a bad structure of the DNA or to a failure of the two recycling systems of the cellular building blocks. Both mechanisms deteriorate with age.

Misfolded proteins can accumulate in the body, even causing the death of some of our cells. This phenomenon is associated with many age-related diseases such as Parkinson’s disease, Alzheimer’s disease … It is therefore considered as one of the primary causes of aging.

The antagonists of aging

These are mechanisms that are initially supposed to protect us, but which eventually become harmful to the body.

5. Mitochondrial dysfunction

Mitochondria are the powerhouses of cells. They maintain cellular respiration and contribute to the essential production of ATP (which provides energy). Mitochondria contain their own genes, the mitochondrial DNA. Abnormalities in these genes are associated with neurological disorders.

The mitochondria produce waste products, the free radicals. These free radicals by oxidation create damage to the mitochondrial DNA and proteins. The oxidative stress process triggers the autophagy mechanism to eliminate the damaged mitochondria.

The second cause that would link mitochondria to aging could be the communication between the nucleus of the cell and its mitochondria. With aging, these communications deteriorate.

6. Nutrient detection pathways

Our organism adapts the behavior of the cells according to the amount of nutrients available to them. Aging alters the process. Nutrient sensing pathways degrade. This can lead to atherosclerosis, the leading cause of cardiovascular disease, which is the leading cause of death in people over 70.

7. The senescence of cells

A senescent cell is an aging cell whose functions deteriorate. It stops dividing and its activity changes. During this change, a normal cell will begin to change its metabolism and may begin to secrete pro-inflammatory molecules that can, in turn, degrade the health of surrounding cells.

Senescent cells can be destroyed by the immune system. But this system decreases in efficiency with age.  Therefore, aging causes senescent cells to accumulate, which in turn will degrade even more surrounding cells.

Secondary causes

9. Stem cell depletion

Stem cells are undifferentiated or partially differentiated cells. While normal cells are only able to divide a limited number of times (the so-called Hayflick limit), stem cells are able to divide in principle indefinitely.

However, with aging, the capacity of stem cells decreases, increasing the number of senescent cells present in the tissues and leading to various problems depending on the organ affected.

10. Inflammation

Inflammation is a complex phenomenon that occurs during an external or internal aggression on tissues. The cells of our body can emit signals according to the stress to which they are subjected. They synthesize molecules called cytokines to regulate the inflammatory response. There are also anti-inflammatory molecules that help regulate the inflammatory response. The goal is to achieve a balance so that the immune response is neither too low nor too high. With age, inflammation becomes more important and the regulations become less effective. This imbalance will cause what is called inflammagging.

Conclusion

The following describes the aspects of aging according to the most accepted categorization today.

There are many other theories related to aging, including the theory developed in Strategies for Engineering Negligible Senescence with not nine, but seven causes.

Does the extreme complexity of senescence mean that we cannot slow aging without addressing all aspects? No, because the causes of aging are very similar at least for many mammalian species. Opinions may vary on this question. However, these species have very different life spans. On the other hand, in order to achieve considerable progress, it is likely that all aspects of senescence will have to be taken into account.

Other text: Clinical trials on humans

Introduction

Aging is a complex and multifactorial process. There are countless theories about why and how aging occurs, and many others claim to be able to stop the aging process and thus increase lifespan.

Genetic approaches to identifying genes that modulate longevity have been very successful, and recent efforts have extended these studies to mammalian aging. The mouse has become the preferred mammalian model. Among the reasons for this choice are its genetic proximity to humans, the possibilities of genetically manipulating its genome, and the availability of many tools, mutants, and inbred strains.  In the field of aging, mice have become very robust and reliable research tools. Studies on transgenic mice have demonstrated that they are useful models for human aging and age-related diseases.  Transgenic mice are mice whose genome has been modified to study the function of certain genes. This modification consists of randomly inserting DNA molecules into the genome of mice.

Another reason why laboratory mice are preferred for research on aging is their short life span, which allows for faster results. Various experiments carried out on mice, as well as numerous genetic interventions, have yielded significant results and have led to a better understanding of the fundamental processes of aging.

In this article, we will review some of the studies that have been conducted on mice, the results that have been demonstrated by these studies and finally, we will consider other areas of mouse research that can be explored by scientists.

Most importantly The Intervention Testing Program (ITP) experiments

Guidelines to Use Animals in Experiments

Multiple rules and regulations must be followed to ensure that the ethics are maintained while using a model organism for experimental purposes. Biological science is constantly evolving; hence, the need to test these on lower organisms before being tested in humans is required in all clinical trials. The EU has a set of strict rules and suggestions that must be followed, these are.

The three Rs-

1.   Replacement- to always replace animal models whenever possible with alternatives like human tissues and cells (Stem cell cultures and Organoids), computer models, and micro-physiological systems. 
2.   Reduction- Have a robust experimental design that can reduce the number of animals that will be involved and produce standard information.
3.   Refinement- To have a refined experiment structure where the pain and suffering of the animal are minimal and provide a humane endpoint.

This humane endpoint is essential in animal ethics and the law requires the animal to not have prolonged suffering under any circumstances (Not even at the cost of the failure of the experiment).

Body Condition Scoring is easily performed by picking a mouse up at the base of the tail, then noting its body condition by passing the finger over the sacroiliac bones (back and pubic bones) and assessing. The Scores are given from 1-5, 1 being the worst which will require immediate euthanasia. In terms of weight loss, a rapid decline of 10-15% of body fat within a few days is a criterion for euthanasia. An overall loss in weight of 20% is also an indication of euthanasia. Other physical indicators like fighting wounds, lack of weight gain after weaning, protrusion of rectum, and any visible tumors and masses should also be observed, and the decision of euthanasia should be taken after an assessment of severity. Other health conditions like anemia, abnormal eye, breathing or head tilt, dehydration, or hypothermia should also be constantly monitored. Not only physical features but also psychological indicators like behavior (active or lethargic), Social or anti-social, and aggressive or timid nature can also indicate the well-being of the mouse and rats.

Rats and Mice at the age of 18 months are ideal to begin experiments related to anti-aging interventions. This is a fairly old age for the animals and they become too frail after this period. 

In conclusion, the veterinary and animal care staff needs to have strong communication and regular and random checks done to ensure that the experimental mice and rats are kept in good condition and the humane endpoint is met when the time comes to ensure minimal suffering by the animals. 

Experiments that have been done and those that remain to be explored.

1. Metformin

Metformin is a drug commonly prescribed to treat patients with type 2 diabetes. It is seen that long-term treatment with metformin (0.1% w/w in diet) starting in middle age extends the health span and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimics some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increases AMP-activated protein kinase activity and increases antioxidant protection, reducing oxidative damage accumulation and chronic inflammation. This study indicates the beneficial effects of metformin on healthspan and lifespan. Metformin is currently under scrutiny for having biased results in the previous studies so it will be important to see the results of the TAME study and confirm if metformin is working for anti-aging.

2.  Rapamycin

Also known as Sirolimus, Rapamycin is a macrolide compound used for preventing rejection after organ transplant, coating stents, and even as a treatment for lung and other types of cancer. It functions by targeting the mTOR which regulates the growth of our cells by binding to a subset in the catalytic cycle, blocking the function of the mTOR.Rapamycin is currently the wonder drug with the most promising anti-aging effects.  The latest studies at the Max Planck Institute for Biology of Aging show that in young adult fruit flies, a 2-week administration of rapamycin can protect them against age-related conditions in the intestine and extend life. Then they administered rapamycin for 3 months in mice and saw similar results with beneficial intestine-related results by the time the mice were middle-aged. This short exposure to the drug is seen to be equally beneficial as lifelong administration with lesser to no side effects. Further studies will now try to answer if the geroprotective effect of rapamycin continues in humans if they start taking it later in life and what kind of dosing will be ideal.

3.  Robust Mouse Rejuvenation

The program aims to achieve “Robust Mouse Rejuvenation” by applying a multi-component intervention to mice of a strain with a historic mean lifespan of at least 30 months. The intervention is initiated at an age of at least 18 months and increases both mean and maximum lifespan by at least 12 months. In each study in this program, the synergy of typically at least four interventions Rapamycin, Senescent Cell Ablation, Telomerase Expression, and Hematopoietic Stem Cell Transplant individually known to extend mouse lifespan when started in mid-life is examined. The ultimate readout of lifespan is determined, as well as the interactions between the various interventions, as revealed by the differences between the treatment groups (receiving different subsets of the interventions) concerning the trajectories with age of cause of death, decline in different functions, etc. In this way, the program adds greatly to the understanding of which benefits these interventions confer and how they synergize or possibly antagonize

4. Resveratrol treatment 

Mice with heart failure when given the resveratrol treatment showed restoration of the mitochondrial oxidative phosphorylation complexes levels. It also restored cardiac AMP-activated protein kinase activation, improved myocardial insulin sensitivity to promote glucose metabolism, and significantly improved myocardial energetic status

Resveratrol, as a SIRT1 activator, extends the lifespan of mice fed a high-fat diet, but has little effect on the mean or maximum lifespan of mice fed a regular diet.SRT1720, a more potent SIRT1 activator than resveratrol, extends lifespan and improves the health span of adult mice fed a high-fat diet or a standard diet. Moreover, SRT1720 enhances insulin sensitivity, improves bone mass, and inhibits tumor growth. Likewise, SRT2104, a first-in-class and highly selective activator of SIRT1, extends the mean and maximal lifespan in mice fed a regular diet, accompanied by improved whole-body physiology. Resveratrol improves osteoblast activity (production of collagenous and noncollagenous proteins) and bone formation. On the other side, resveratrol could extend the health span in aging rodents]. Resveratrol treatment seems safe and well-tolerated in phase II clinical trial,

5. Glucosamine 

Promotes longevity by mimicking a low-carb diet, a study finds The widely used food supplement glucosamine promotes longevity in aging mice by approximately 10 percent due to improved glucose metabolism. Researchers find that the compound does so ‘by mimicking a low-carb diet in elderly mice reflecting human retirees.’ In addition, the study’s results seemed to indicate protection from diabetes, a life-threatening disease most prevalent among the elderly.

6. Fecal Microbiota Transplant From Young Mice Improves Muscle and Skin in Old Mice

The gut microbiome changes with age, and some studies show that this shift can lead to degenerative aging. An increase in inflammatory microbes and microbes producing harmful metabolites with fewer microbes generating beneficial metabolites is seen. One of the methods is to transplant fecal matter from a younger into the gut of an older individual. The results from a study show that the young-derived gut microbiota rejuvenates the physical fitness of the aged by altering the microbial composition of the gut and gene expression in muscle and skin. Dbn1 (cytoplasmic actin-binding protein) for the first time, was found to be induced by the young microbiota and to modulate skin hydration. 

7. New intranasal and injectable gene therapy 

Previous studies showed that the adeno-associated virus (AAV) vector induced overexpression of certain proteins, which can suppress or reverse the effects of aging in animal models. In our study, we sought to determine whether the high-capacity cytomegalovirus vector (CMV) can be an effective and safe gene delivery method for two such protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). This study conducted by collaboration between George Church and Elizabeth L Parrish found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. They show CMV being successful as both an intranasal and injectable gene therapy system to extend longevity. This treatment also significantly improved glucose tolerance, and physical performance, as well as prevented body mass loss and alopecia. Further, telomere shortening associated with aging was ameliorated by TERT, and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with quality of life and an increased health span.

8. Blood Dilution

Heterochronic blood sharing rejuvenates old tissues, and most of the studies on how this works focus on young plasma, its fractions, and a few youthful systemic candidates. This study by Irina M. Conboy and her team recently developed a small animal blood exchange process. They replaced half of the plasma in mice with saline containing 5% albumin (terming it a “neutral” age blood exchange, NBE) thus diluting the plasma factors and replenishing the albumin that would be diminished if only saline was used. The data demonstrate that a single NBE suffices to meet or exceed the rejuvenating effects of enhancing muscle repair, reducing liver adiposity and fibrosis, and increasing hippocampal neurogenesis in old mice, all the key outcomes are seen after blood heterochronicity. Comparative proteomic analysis on serum from NBE, and from a similar human clinical procedure of therapeutic plasma exchange (TPE), revealed a molecular re-setting of the systemic signaling milieu, interestingly, elevating the levels of some proteins, which broadly coordinates tissue maintenance and repair and promoting immune responses. Moreover, a single TPE yielded functional blood rejuvenation, abrogating the typical old serum inhibition of progenitor cell proliferation

9. Effect of Young Rat Plasma on Lifespan of Aging Rats

A study on older rats to test longevity after plasma transfusion of young rats (9 tested old rats + 8 control old rats). Testing begins in November 2020. This experiment is under the direction of Professor Rodolfo Goya at the Institute of Biochemical Research in Argentina, in collaboration with Heales. 

The results showed that young plasma treatment in old rats increases their lifespan by 2.2 months, and their external appearance is healthier than that of untreated rats. Moreover, the young plasma treatment resulted in consistently lower epigenetic age in treated rats compared to untreated ones. However, no significant differences were detected between the control and treated groups. On the contrary, there was a significant difference in DNAm age between control and treated groups within the age ranges of 27-31.5 months. The analysis of differentially methylated CpGs showed that the plasma treatment induced DNA methylation modifications in 1.6% of all rat CpGs. Additionally, the differentially hypomethylated promoters were associated with insulin-like growth factors (IGF) related gene promoters, while the differentially hypermethylated promoters were associated with chemo and cytokine gene promoters. Finally, when rats were grouped according to the similarities in their differential blood DNA methylation profile through hierarchical clustering, samples from the treated and control rats were clustered in separate groups.

10. Effect of Elixir Plasma on Lifespan of Aging Rats (E5)

A study on older rats to test longevity after plasma fraction with the working name ‘Elixir’ injection into old rats (6 experimental old rats + 6 control old rats). Testing started in December 2020. The experiment is under the direction of Professor Harold Katcher in Mumbai, in collaboration with Heales

A recent preliminary study reports by Harold Katcher that repeated intravenous administration (an effective alternative to parabiosis) of a plasma fraction (called Elixir) from young rats to aged counterparts for 5 months, changes the epigenetic age of the liver, blood, and heart tissue of the old treated rats (25 months) to an age close to that of young adult rats (7 months). This apparent rejuvenation was confirmed by Steve Horvath’s DNA methylation clocks.

Based on the above information, we decided to evaluate the possible effect of “Elixir” on the lifespan of older (25 months) rats. Specifically, we propose to compare the survival of old rats treated intravenously with young plasma with that of correspondingly aged (untreated) controls. We also propose to collect blood samples from all animals, every other week, to follow the evolution of epigenetic age over time.

The study used grip strength to assess age-related decline in muscle function and motor coordination. The treated group showed a significant improvement in grip strength compared to the control group. The treatment improved muscle coordination functions by 2-3 times as measured by the grip strength meter. The study also monitored the mortality rate of the rats. The first death occurred in the control group at 33.99 months, and subsequent deaths were seen at the ages of 35 and 37.89 months. The maximum age reached in the control group was 39.49 months. In the treated group, the first death occurred at 38 months, with mortality notes between 39 and 41 months. The treated group’s average longevity increased, reaching 48 months of age.

This study suggests that E5 treatment increases antioxidant markers and grip strength of treated animals while reducing cytokines concentration and improving the integrity of the vital organ, ultimately increasing the lifespan. However, further information and research are required to determine the precise nature of the variables present in the E5 and to confirm the results with a bigger group of animals.

11. Mitochondrial uncoupling 

In a study, researchers show that increased endogenous, uncoupling protein (UCP1) mediated, as well as experimentally induced mitochondrial uncoupling to an increased lifespan in rodents. This is possibly due to the synergistic activation of molecular pathways linked to the life-extending effects of caloric restriction as well as a mitohormetic response. Mitohormesis is an adaptive stress response through mitonuclear signalling which increases stress resistance resulting in health-promoting effects. Part of this response is the induction of fibroblast growth factor 21 (FGF21) and growth and differentiation factor 15 (GDF15), two stress-induced mitokines that elicit beneficial systemic metabolic effects via endocrine action.

12. Mitochondrial biogenesis

Perturbed mitochondrial function has been correlated with severe human pathologies such as type-2 diabetes, and cardiovascular, and neurodegenerative diseases. Thus, proper mitochondrial physiology is a prerequisite for health and survival. Cells have developed sophisticated and elaborate mechanisms to adapt to stress conditions and alterations in metabolic demands, by regulating mitochondrial number and function. Hence, the generation of new and the removal of damaged or unwanted mitochondria are highly regulated processes that need to be accurately coordinated for the maintenance of mitochondrial and cellular homeostasis.

13. Spermidine targeting autophagy activation

The natural polyamine (proliferation of neoplasms in the gastrointestinal tract) spermidine extends the lifespan of mice and exerts cardioprotective effects in old mice via autophagy activation. Autophagic rates decline with age in most organisms, as a potential mechanism underlying many age-related pathologies, like Parkinson’s and Alzheimer’s diseases. Indeed, polyamine synthesis decreases with aging and the boosting spermidine level by spermidine intake or gut bacteria-produced polyamine is capable of lifespan promotion in short-lived mouse models. Life extension of up to 25% can be produced by lifelong spermidine administration, accompanied by reduced liver fibrosis and hepatocellular carcinoma. It is also seen that spermidine activates the casein kinase 2 (CK2), ameliorates aging features, and extends lifespan in a mouse model of HGPS.

14. Gene editing using CRISPR-Cas9 technology

Researchers in recent times have successfully used a DNA-editing technique to improve the lifespan of mice with the genetic variation associated with progeria, a rare genetic disease that causes extreme premature aging in children and can significantly shorten their life expectancy.

In another study, scientists found that CRISPR-Cas9-mediated disruption of the mutant HTT gene resulted in a ∼50% decrease in neuronal inclusions and significantly improved lifespan and certain motor deficits. These results thus illustrate the potential for CRISPR-Cas9 technology to treat HD and other autosomal dominant neurodegenerative disorders caused by trinucleotide repeat expansion via in vivo genome editing.

15.  Caloric Restriction

Caloric restriction (CR), is one best-studied method to improve quality lifespan in most organisms, as it targets and regulates pathways like the kinase target of rapamycin (TOR), AMP-activated protein kinase (AMPK), sirtuins, and insulin/insulin-like growth factor. CR counteracts the aging process by regulating a set of evolutionarily conserved pathways, 

CR alone could reduce seizure susceptibility in epileptic mice. It is important to mention that CR in the mouse is not the same physiologically as CR in humans since the basal metabolic rate is about seven times greater in mice than in men. CR in mice is similar to therapeutic fasting in humans since a 40% CR in inbred C57BL/6J mice produces changes in serum lipids similar to those seen in humans following therapeutic fasting or very low-calorie dieting (below 500 kcal day−1). Hence, CR in mice can be an effective model system for investigating the anticonvulsant and antiepileptogenic mechanisms of therapeutic fasting in humans.

16. Exercise

To address factors underlying aging due to a decline in mitochondrial function, and the effects of exercise during aging, this study went ahead to profiled proteins in the brain and muscle of sedentary and exercised mtDNA (Mitochondrial DNA)  mutator mice. The results showed that voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In-depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear–mitochondrial crosstalk. These results indicate that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.

Conclusion

 The road to reaching our goals is long. Efforts in all directions are to be considered and promoted. Studies with rats have provided us with significant lessons for our research of all kinds. According to an article by Mari Shimoyama, rats remain a major model for the study of disease mechanisms and the discovery, validation, and testing of new compounds to improve human health.  At Heales, we encourage and invite more investors to invest in and promote research on rats to unlock the secrets of aging.

One of the main challenges of research for healthy human longevity is to organize reliable studies (double-blind…) and to publish the results (even if they are not positive).

This page gives a list of what the organization Heales considers the most important and promising clinical trials that have already been started or should be started as soon as possible. As the studies are in progress, it is important to keep ourselves updated on the latest news which included criticism from the scientific community.

This list is intended to be a starting point. You are more than welcome to comment or make other suggestions.

An article about trials on animals will be published later.

• TRIM study Thymus
• Metformin TAME Study
• Rapamycin
• Senolytics
• NAD+ 
• Plasma dilution 
• Telomere
• Muscles Folystatin 
• Alpha-Ketoglutarate (AKG)
• Khloto

1. TRIM study Thymus

The thymus is a primary lymphoid organ essential for the development of T cells. These T cells are majorly responsible for adaptive immunity or the first line of defense against bacteria, viruses, and also tumors in the body. As time passes, the thymus goes under programmed cell atrophy known as involution. All T cells are produced at a young age as the thymus is at its largest size in children. As people grow older, the thymus starts to shrink and involution especially hits the immune system of people between the ages of 65 and 75. Thymus Regeneration, Immunorestoration, and Insulin Mitigation Extension Trial (TRIIM-X) by scientists at Intervene Immune were originally run from 2015 to 2017 on participants from age 50-65 that are healthy men that received a combination of growth hormone DHEA and metformin with an objective of reversing thymic involution. The results were promising they show that the participants showed an increase of more than two years in the predicted human lifespan. This was seen throughout the various epigenetic clocks including the GrimAge clock that analyzes DNA methylation. As of today, they have started another trial with similar objectives and are currently looking for volunteers. FDA has not approved this therapy yet but TRIIM-X Trial was the first of its kind to show regression in multiple age-related biomarkers possible in humans.

2.  Metformin TAME Study 

First introduced in 1922, Metformin is a well-known drug given to patients with type 2 diabetes and is known to act by helping the body to reabsorb less sugar and hence balance the blood sugar levels. It both lowers glucose absorption after a meal, as well as increases the body’s sensitivity to insulin. It is a popular medicine as it is cheap in cost and has minimal side effects. Metformin has already shown signs of delaying aging The main aim of the TAME (Targeting age with Metformin) Trial is to study how metformin delays age-related diseases like CVD, neurological conditions, cancer, etc. Led by Dr. Nir Barzilai, the trial is set to be six years long with more than 3000 participants from the age 65 to 79. Their goal is that if the FDA declares ‘aging’ itself as a disease, then instead of targeting the different age-related conditions, they will target aging itself which will include all the conditions together.Along with the TAME trial, they also want to look for biomarkers of aging in TAME BIO by:

• Store samples of the blood, urine, DNA, etc from all the participants to study further.
• Use different approaches to study biomarkers that predict changes in function, multi-morbidity, and subsequent death.
• Study the influence of Metformin on multiple age-related biomarkers on all participants using and create a report with the core data collected.

They are currently collecting funds to proceed with the trial as the TAME Trail team deeply believes that drug intervention for aging and related diseases will not only extend years of health but will also save trillions of dollars. 

3. Rapamycin

Also known as Sirolimus, Rapamycin is a macrolide compound used for preventing rejection after organ transplant, coating stents, and even as a treatment for lung and other types of cancer. It functions by targeting the mTOR which regulates the growth of our cells by binding to a subset in the catalytic cycle, blocking the function of the mTOR.

Rapamycin is currently the wonder drug with the most promising anti-aging effects.  The latest studies at the Max Planck Institute for Biology of Aging show that in young adult fruit flies, a 2-week administration of rapamycin can protect them against age-related conditions in the intestine and extend life. Then they administered rapamycin for 3 months in mice and saw similar results with beneficial intestine-related results by the time the mice were middle-aged. This short exposure to the drug is seen to be equally beneficial as lifelong administration with lesser to no side effects. Further studies will now try to answer if the geroprotective effect of rapamycin continues in humans if they start taking it later in life and what kind of dosing will be ideal.

4. Senolytics

Senolytics come under a class of drugs that clears out senescence cells (SC). Senescence is a phenomenon where the cells get arrested in the cell cycle if they sense danger stimuli. Accumulation of the SC increases with aging and can cause damage to the tissue and can be a cause of multiple diseases including various neurodegenerative diseases. 

Dasatinib (a tyrosine kinase inhibitor), Quercetin (a naturally occurring flavonoid), Fisetin, and Navitoclax were the first senolytic-drugs introduced in the market following a hypothesis-driven approach. A combination of Dasatinib and Quercetin was given to mice for over two years and the results showed fewer senescence-related biomarkers as well as a lower occurrence of disc degeneration. However, this result was seen in young and middle-aged mice, not the older ones. The challenge currently lies in the lack of biomarkers specific to senescent cells. Currently, SA-β-Gal is the only available biomarker. Poor drug specificity delivery system is another challenge that the researcher needs to work out. More than 20 different clinical trials are being held to improve the efficiency of senolytics and the results from these will form a promising future.

5. NAD+ Charles Brenner

Nicotinamide adenine dinucleotide (NAD) is a coenzyme for redox reactions and is responsible for transferring electrons during metabolic processes. According to the study of Dr. Chareles Brenner, Nicotinamide riboside (NR) goes into the cells and nucleoside kinase puts a phosphate on by which NR is turned into NMN (Nicotinamide mononucleotide). NMN then converts to NAD and finally to NADP

6. Plasma dilution 

It is a method where the blood from an individual is extracted and then a plasma fraction of that blood is replaced by saline and albumin and this new mix is reintroduced in the body. Plasma dilution, also known as neutral blood exchange, is said to have properties that can restart the body’s regenerative capacities. 

The latest clinical trial from the University of California shows that Therapeutic plasma exchange (TPE) has the potential to restore the younger system and reduce biological aging. This study had a very small group of participants (Only 8) but has promising results with the de-regulation of 10 novel biomarkers, a youthful shift to various age-related conditions showing apoptotic regulators, and a youthful profile of myeloid/lymphoid markers in circulating cells, which have reduced cellular senescence and lower DNA damage. Further studies with the biggest participant group will confirm these results to be favorable in reducing aging.

7. Telomere

According to the Hayflick limit, we know that human cells will replicate for only a limited time and this was confirmed when scientists discovered that telomers shortens after each replication. Telomere has the function of protecting the ends of the genome from degradation and maintaining its integrity of it. Telomerase is the enzyme that catalyzes the extension of the telomeric region and the shortening of this telomeric region is associated with signaling between somatic cells for senescence. 

Shortening of telomere with age is related to factors like apoptosis, and oncogenic transformation and even affects the life and health span. The TACTIC (Telomerase ACTivator to reverse Immunosenescence in Acute Coronary Syndrome) trial from the UK had the objective to check if TA-65MD, a telomerase activator can decrease reduce the shortening of telomerase and oxidative stress.90 Patients took 1 x capsule twice daily for 1 year which may be either the test drug TA65MD (8mg) or the place. Another Clinical Trial from France Telomeres and Arterial Aging (TELARTA). Their objective is to create a model that makes it possible to examine different elements of telomere length dynamics in different tissues: leukocytes, skeletal muscle, endothelial progenitor cells (EPCs), and skin or subcutaneous fat in patients with or without atherosclerosis. The results from these and many such trials will give us a better view of future treatments. 

8. Follistatin 

Activin binding protein, follistatin, is encoded by the FST gene in humans and it is an autocrine glycoprotein, seen in most tissues. It is an antagonist of myostatin involved in increasing strength and muscle mass. This is one of the reasons why follistatin supplements are popular amongst bodybuilders.  

Follistatin gene therapy is emerging and Bioviva CEO (in 2015) became the patient zero for the therapy of both telomerase (hTERT) and follistatin (FST). A study using Mouse mouse cytomegalovirus (MCMV) carrying exogenous FST has shown an increase of 32.5% in median lifespan. Another study targeting Sarcopenia (loss of muscle mass and strength over age) showed overexpression of follistatin delivered by Adeno-associated virus (AAV) in mice aged 24-27 months caused an increase in muscle weight as well as improvement in age-related degeneration at the neuromuscular junction in mice.

9. Alpha-Ketoglutarate (AKG)

AKG is a ketone derivative of glutaric acid which is known to be used by the growing cells and is involved in the healing of wounds and injury (especially muscle tissues). As AKG is an endogenous intermediary metabolite in the Krebs cycle whose levels naturally decline during aging, many supplements are currently available for the public. Rejuvant, is a  potential life-extending compound formulation with alpha-ketoglutarate and vitamins, which has been shown to have an average of 8-year reduction in biological aging, after an average of 7 months of use, in the TruAge DNA methylation test. 

10. Khloto

Klotho is a transmembrane protein that is involved in the regulation of oxidative stress, signaling growth factors, organ protection, and control over the sensitivity of organisms to insulin and shows involvement in the aging process. 

Current studies show that higher Klotho levels in an 85-year-old will have the same size of brain and the thinking ability to that as someone who is 10 years younger. Another study shows that a 20%-30% increase in the lifespan of animals was observed after the overexpression of Klotho. With a single injection, Klotho showed improved cognition by 2 years in terms of spatial and working memory. Klotho Therapeutics is one of the leading biotech firms which strongly believes in Klotho having anti-aging properties and developing a patent-pending Klotho protein that has great potential to redefine society’s experience with aging.