Longevity Cookbook: Combinations of Life Extending Drugs

Here is a teaser from the Longevity Cookbook project. The first chapter is on pharmacologic enhancement of lifespan. This chapter includes different ways of trying to develop pharmaceuticals to combat aging. What are the different research avenues? Are there special considerations when trying to develop treatments for aging? What are the pitfalls? It also includes an overview of the field as it stands today. Different drugs or substances that are especially promising or interesting are discussed. Can we meaningfully impact healthy lifespan through pharmacological means?

The second part is an ambitious proposal for testing a mixture of promising compounds for their effect on the health and lifespan of mice. This includes six compounds tested individually side by side for their effects on longevity and a variety of health-span measures. We will also try to determine the optimal dose and see if the compounds together have an additive or even possibly synergistic effects on lifespan.

We hope you enjoy it, and if you do, there is much more coming in the Longevity Cookbook.

Project: Preclinical study of longevity-promoting drug combinations

Introduction

Genetic studies performed in animal models (yeast, nematode, Drosophila, mouse) have demonstrated the regulatory role of intracellular signaling pathways (insulin/IGF, FOXO, JNK, TOR) in the lifespan control [1]. Evolution has changed animal’s lifespans over 2000 fold. We also know we can affect lifespan with pharmacueticals[2–4]. Some of these will affect these signaling pathways and some may interfere directly in detrimental processes such as glycation. Each has some effect on lifespan. What is not well studied is how these interventions work together.

In this study we selected 7 pharmaceuticals to test their effect on lifespan and health span in combinations. All these substances affect some aspect of aging, and all of them are used in clinical practice. Their optimal doses for the purpose of extending lifespan and health span are not known and how they will work together is what we will determine.

Description of the selected longevity-promoting substances

1. Metformin is used for the treatment of type 2 diabetes. In experiments on C57BL/6 mice metformin increased their life expectancy by 4–6% and improved a number of age-related indicators, such as physical activity and insulin sensitivity, lowered low-density lipoprotein and cholesterol levels [3]. In addition, clinical studies in humans with type 2 diabetes demonstrated that metformin treatment leads to a statistically significant increase in survival as compared with untreated patients of the same age (non-diabetic people) [5]. Metformin also reduces risk of cancer [6] Metformin affects an enzyme AMPK, which triggers stress response, reduces reactive oxygen species levels and delays aging,[7]. Moreover, metformin is a non-enzymatic glycation inhibitor.
2. Deprenyl (selegiline) is a monoamine oxidase (MAO) type B inhibitor. Numerous experiments on rodents and even elderly dogs significantly extended their lifespan (including maximum lifespan) [8–10].
3. Glucosamine is an amino sugar, which is produced by cartilaginous tissue in human organism and is a part of the cartilage. Glucosamine treatment increased nematode lifespan by approximately 5% and the life expectancy of 100-week mice by about 10% (with a slight increase in the maximum lifespan)[11]. In human studies glucosamine resulted in reduction of mortality from cancer (13%), respiratory diseases (41%) and other reasons (33%). Glucosamine inhibits inflammation affecting cytokines and NF-kB [12] It is also a mimetic of low-carbohydrate diet[13].
4. Rapamycin Inhibits mTOR and shows robust positive effects on lifespan in a range of model organisms[15].
5. Aspirin A nonsteroidal anti-inflammatory drug increases lifespan in male mice [4] and reduces mortality and cancer in humans [16].
6. Nicotinamide mononucleotide (NMN) is a NAD precursor. NAD is one of the most important compounds necessary for normal cell functioning, as it is an electron transporter and a substrate of numerous enzymes sirtuins, which have shown lifespan extending effects. NMN introduction to 22-month old mice reversed pseudohypoxic state of cells, which is an important feature of aging [12].

Project goal: To examine if a combination of life extending substances can significantly extend life and improve health over and above the effects of the single substances alone. Determine the optimal dose and study possible toxic interactions or synergies.

Objectives:

Test longevity-promoting effects (changes in lifespan, dynamics of body weight, physical activity, cognitive function, complete blood count and frailty) of 6 drug combination (glucosamine, rapamycin, aspirin, nicotinamide mononucleotide, metformin, deprenyl) in C57BL/6 18 months aged mice.

We will be able to determine if these compounds work well in combination, and if so at which dose. If the combination does not yield a significant improvement in lifespan, we will still get plenty of data on optimal doses of these compounds. Using a sample size of 20 males and 20 females in the group will make it difficult to separate out small differences in lifespan between the treatments. However, we will be taking longitudinal, clinically relevant measurements of each mouse. This will increase our statistical power greatly and we will be able to determine the effect on the health of the mice.

As a result of the project the most comprehensive database of all longevity-promoting substances is created. It includes their effects on lifespan, activity, stress resistance, molecular targets, side effects of used doses and toxicity. Analytical mathematical models will be developed to cluster and rank the most effective pharmaceuticals, as well as to predict new potential life extending drugs.

The project is implemented taking into account the following statements:

- Chemically pure substances are used as active ingredients;

- Animals will be assessed before the experiment begins to generate a clinically relevant frailty index (FI). This includes assessment of 31 outwardly visible parameters such as fur loss, cataracts and kyphosis according to[17]. These measurements will then be collected every 3 months to generate longitudinal data of the decline or improvement;

- During experiment cognitive functions (Morris test) of experimental animals, as well as their physical activity (“Open Field”) are tested;

- During experiment blood will be collected to measure the level of compound in the blood and total blood count;

- Record of animal survival is performed using modern statistical methods and analytical modeling.

20 male and 20 female C57BL/6 mice aged 18 months are used in each experiment version (group). This is roughly powered to detect a 15% difference in lifespan if there are no sex specific effects, and a 20% difference if effects are dependent on sex.

The experimental scheme is given in table 1.

Notes to table 1:

In experiments all the substances are used in following base doses:

1. Metformin: 1g/kg in chow is accepted as a base dose [3]
2. Deprenyl (selegiline): 0.25 mg/kg injected 3 times/week is accepted as a base dose [10]
3. D-Glucosamine: 10 g/kg in chow is accepted as a base dose [11]
4. Rapamycin 42mg/kg in chow as base dose [2]
5. Aspirin 20mg/kg in chow as base dose [4]
6. Nicotinamide mononucleotide (NMN): 500 mg/kg/day intraperitoneal injection as a base dose[18]

In addition to studies of a base dose of drugs, the effect of one lower and one higher dose will be tested (1/3, 2X of base dose) is tested as well. Lower doses are tested in order to reduce the overall load on the kidneys and liver, which is particularly important in case of longevity-promoting therapy. With the same purpose in case of intervention number 2 pauses in treatment are made.

To study each dose effect a separate group of animals is required. Due to possible differences of treatment effect on health and longevity for males and females, each intervention is performed on male and female group. Thus, to test the therapeutic effects of each pharmaceuticals combination (№№ 1–6) 6 experimental groups are required.

We invite you to form a nonprofit organization working on testing of different combinations of compounds. We believe that anti-aging drugs should be developed using an open non commercial approach, not limited by IP protection and available for everyone.

By Mikhail Batin, illustrated by Olga Posukh

Disclaimer

This is not intended as a guide to health and longevity. It is meant to illuminate some of the interesting findings in this field. We do not recommend any of these substances. You should consult your physician before trying any of this. Do not order these substances online from unverified sources there is no way to know what you are getting without substantial laboratory analysis. We would also not advise taking several substances in combination. Even less is known about the effects of combinatorial interventions. We hope to learn what types of therapies are effective and in the future be able to make recommendations.

Literature:

1. Kenyon, C.J., The genetics of ageing. Nature, 2010. 464(7288): p. 504–12.

2. Miller, R.A., et al., Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell, 2014. 13(3): p. 468–77.

3. Martin-Montalvo, A., et al., Metformin improves healthspan and lifespan in mice. Nat Commun, 2013. 4: p. 2192.

4. Strong, R., et al., Nordihydroguaiaretic acid and aspirin increase lifespan of genetically heterogeneous male mice. Aging Cell, 2008. 7(5): p. 641–50.

5. Bannister, C.A., et al., Can people with type 2 diabetes live longer than those without? A comparison of mortality in people initiated with metformin or sulphonylurea monotherapy and matched, non-diabetic controls. Diabetes Obes Metab, 2014. 16(11): p. 1165–73.

6. Bowker, S.L., et al., Glucose-lowering agents and cancer mortality rates in type 2 diabetes: assessing effects of time-varying exposure. Diabetologia, 2010. 53(8): p. 1631–7.

7. Onken, B. and M. Driscoll, Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1. PLoS One, 2010. 5(1): p. e8758.

8. Kitani, K., et al., Chronic treatment of (-)deprenyl prolongs the life span of male Fischer 344 rats. Further evidence. Life Sci, 1993. 52(3): p. 281–8.

9. Ruehl, W.W., et al., Treatment with L-deprenyl prolongs life in elderly dogs. Life Sci, 1997. 61(11): p. 1037–44.

10. Knoll, J., The striatal dopamine dependency of life span in male rats. Longevity study with (-)deprenyl. Mech Ageing Dev, 1988. 46(1–3): p. 237–62.

11. Weimer, S., et al., D-Glucosamine supplementation extends life span of nematodes and of ageing mice. Nat Commun, 2014. 5: p. 3563.

12. Gomes, A.P., et al., Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell, 2013. 155(7): p. 1624–38.

13. Bell, G.A., et al., Use of glucosamine and chondroitin in relation to mortality. Eur J Epidemiol, 2012. 27(8): p. 593–603.

14. Harrison, D.E., et al., Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature, 2009. 460(7253): p. 392–5.

15. Rothwell, P.M., et al., Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet, 2011. 377(9759): p. 31–41.

16. Whitehead, J.C., et al., A clinical frailty index in aging mice: comparisons with frailty index data in humans. J Gerontol A Biol Sci Med Sci, 2014. 69(6): p. 621–32.

17. Yoshino, J., et al., Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab, 2011. 14(4): p. 528–36.