• MetforminPharma

The pharmacology of metformin, the new anti-aging drug

Introduction

Metformin is currently the most prescribed and used drug in the world against type -2 diabetes or non-insulin-dependent diabetes mellitus (NIDDM) (Dunn and Peters, 1995).

Metformin is a derivative of biguanidin (Figure 1). It has been used to replace another biguanidin molecule called phenformin which causes a higher risk of mortality (Dunn and Peters, 1995). As a safer and relatively cheap drug, its use has been exponentially increased from 1980s (Vecchio et al, 2014). Now, it is known as the first line of prescription. It is marketed under the trade name Glucophage or its generics. Metformin oral tablet is the most common drug form (Figure 2) (Dunn and Peters, 1995).

In addition to its anti-diabetic effects, other positive effects were reported by different studies on laboratory animals (Barzilai et al, 2016). It could reduce the effects of age and increase the lifespan . These properties are very interesting for the scientific community. So, a new project called “Targeting Aging with Metformin” (TAME) is in progress (Barzilai et al, 2016). Its goal is to study the link between metformin and its potential anti-aging effects on humans.

In this essay, the common anti-aging effects are described. Then, the current consensed mechanism of action of metformin is presented. Finally, the pharmacovigilance including the most observed side effects of this drug is reported.

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The diverse positive effects of metformin on the life span and health span

Metformin and its effects on lifespan of different animal species

Positive dose-dependant effects of Metformin on the lifespan of worms

Onken and Driscoll (2010) studied the effects of metformin therapy on nematodes species C elegans. The concentration of 0.00001 to 1.0 mg/l has been applied starting from the larval stage of the nematode untill its whole life . Metformin added at 50 mM dose was shown to increase only the mean lifespan (not the maximum lifespan). A supplementation of concentration of 10 or 100 nM doses didn’t had any significant impact on the lifespan of the C elegans. Anisimov (2013) similarly reported that metformin therapy at doses 25nM increases mean lifespan by 18%, at 50 nM the increase is by 36% and at 100 mM by 3%.

Thanks to these studies, it was shown that metformin given at a certain concentration prolongs nematode health span, extends their mean lifespan and prolongs their youthful locomotor ability.

Effects of metformin on the lifespan of different mice models

Postive effects of metformin on female mice with tumors predisposition

Studies were made on female transgenic HER-2/neu group of mice which is a mice model expressing tumors (indeed this strain of mice mostly develop spontaneous mammary tumors, pulmonary and lymph node metastases). While comparing HER-2/neu group to a control group it has been shown that the supplementation of metformin slowed down the age-related rise of blood glucose, the triglyceride and cholesterol levels and the age-related irregularity in the estrous cycle. Furthermore, it reduced the level of β-lipoproteins. It extended the mean lifespan by 4-8% and the maximum lifespan by 1 month. In addition, it normalized the expression of cytolytic granzyme B and perforin gene in malignant mammary tumors (Anisimov, 2010).

Age-dependant metformin effects on SHR female mice

A study on female SHR mice (acronym for Spontaneously Hypertensive Rat, which are rat with hypertension and used to study cardiovascular diseases) showed that a supplementation with metformin increased the mean lifespan of the survivors by 20% and their maximum lifespan by 10.3% (Anisimov, 2013). Metformin treatment delayed the age-related irregularity of the estrous function. However, no effect was observed on the following points : serum level of cholesterol, triglycerides, glucose, insulin and carcinogenesis. Another study based on different set of age of SHR mice showed that Metformin given at age 3, 9 and 15 months increased the mean lifespan by respectively 14%, 6% and 0% (Anisimov, 2010).

Sex-dependant effect of metformin on 129/Sv mice and trangenic mice with Huntington disease (HD)

In 129/Sv mice as animal model test, a supplementation of metformin of 100 mg/kg induced a modification of their food intake but didn't change their body weight. Applied on male 129/Sv mice, a decrease of the lifespan of 13.4% and no decrease of spontaneous tumor development were observed. On the contrary, an increase of 4.4% of the mean lifespan and a decrease of the spontaneous tumor development were reported on female 129/Sv mice (Anisimov, 2010). Other studies were made on transgenic mice with Huntingon disease, which is a neurodegenerative disease that leads to striatal degeneration and severe movement disorder. Metformin treatment increased the lifespan on male mice by 20.1%. However, no increase were observed on female mice receiving the same treatment (Anisimov, 2013).

Dosage-dependant effect of Metformin on male C57BL/6 and B6C3F1 mice

Male C57BL/6 mice (which is the most used laboratory rodent) supplemented by 0.1% of Metformin on diet ad libitum had an increase of 5.83% of their lifespan. A supplementation of 1% of Metformin leads to a decrease of 14.4% of their lifespan (Ani et al, 2013). The same experiment on B6C3F1 mice resulted by an increase of the lifespan by 4.15% on a ad libitum diet of 0.1% of metformin (Anisimov, 2013). The anti-ageing effects of metformin depends on the dosage and on the strain of the mice.

General effects of Metformin observed on mice

In all the previously mentioned studies, some particular beneficial effects were pointed out such as improved physical performance, delayed metabolic syndrome such as improvement of glucose tolerance, increase of the insulin sensitivity, a decrease of the LDL ( Low Density Lipoprotein) and cholesterol level. These positive effects depends on the stain of the mice, on the sex, on the onset of the treatment and on the dosage of metformin.

No significant or toxic effect on the increase of lifespan of F344 rats and Drosophila

• 6 months old male F344 rats were divided in 4 groups : a control group, a calorie restricted group, a metformin supplemented group and pair fed to metformin (Anisimov, 2010). All group presented no significant differences concerning their mean lifespan and the mean age of the last surviving 10%. The researchers suggest that the effect of metformin is similar to the effect of calorie restriction. In a similar way, independent studies on male and female Drosophila have shown that metformin supplemented in doses 0.4, 0.8, or 0.16 mg/ml doesn’t influence their mortality rate (Anisimov, 2010). No significant effect on their survival has been shown. A significant decrease of their survival were even observed at the maximal dose. Consequently, metformin may have no significant or a toxic effect on the lifespan on some animal species like F344rats or Drosophilia.

Conclusion on the effect of Metformin on laboratory animals

These study on animals showed that Metformin therapy has generally a positive dose-dependant effect on lifespan and healthspan in mice and worms. Indeed, metformin can be toxic above a species-dependant level. Metformin therapeutic dosage is species and strain specific.

Metformin therapy reduces the risk of age-related cancer

Study of the effects of metformin on ovarian cancer patients with diabetes

Metformin reduce the diabete type 2 and consequently it reduces the risk of cancer. A study on 568 consecutive patients, newly diagnosed with ovarian cancer including patient with ovarian, fallopian or peritoneal cancer and excluding patients with type 1 diabetes, incomplete medical records and patient with any other cancer before their ovarian cancer diagnosis. 48 (8.5%)of these 568 patients with type 2 diabetes received a continuous treatment of metformin, 34 (5.9%) patients with type 2 diabetes were part of the diabetic control group(no metformin treatment), 22 (3.9%) patients with type 2 diabetes received a discontinued metformin treatment, and 464 (81.7%) ovarian cancer patients were part of nondiabetic controls. The results showed that the group taking continuous metformin treatment had a longer stabilization of the tumors (progression-free survival) and a better overall survival (OS) compared with the 3 others groups (P = 0.001). Furthermore, patients taking continuously Metformin had a lower risk for disease relapse death [ P= 0< .01] and disease-related death (P= .03) ( Wang et al, 2017). Consequently it’s required that the Metformin treatment has to be continuous in order to have beneficial effects such as a reduction of the risk of disease recurrence and a reduction of risk of death in ovarian cancer patients having diabetes.

2.2.2.2 Effects of metformin on cancer occurence and mortality

According to statiscal studies (Table 1), metformin treatment is associated to the reduction of the risk of cancers. It significantly reduces the occurence of the most frequent and severe age-related cancers such as colorectal, liver, pancreatic, stomach or oesophagus cancer. Consequently, metformin is a very interesting potential anti-tumor drug.

* : pvalue <0,05 ; ** : pvalue<0,01 ; *** : pvalue < 0.001.

Table 1. Effects of metformin on different types of cancer. (Adapted from Franciosi et al, 2013).

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Mechanism of action

Therapeutical dosage recommandations

Metformin was shown to reduce visceral adiposity and insulin resistance after 8 weeks of drug therapy at dose of 850 mg, 3 times per day (Saint-Marc and Touraine, 1999).

Pharmokinetics of metformin

Bioavailability

The oral bioavailability of metformin is 55%± 16% (mean ± SD). No or low protein-binding have been reported. It is absorbed mainly by the small intestine (Graham et al, 2011).

Metabolism and Excretion

Metformin is excreted unchanged by urine. The half-life of its elimination during multiple dosages with normal renal function is about 5 hours (Graham et al, 2011).

Membrane-Transporters of Metformin

The pharmacokinetics of metformin is mainly determined by membrane transporters (Table 2).

Table 2. Membrane-transporters of metformin. (Adapted from Chen et al, 2013 ;Gong et al, 2013).

The pharmacologic effects of metformin are primarily exerted in the liver, at least partly via the activation of AMPK and the subsequent inhibition of gluconeogenesis (Chen et al, 2013; Gong et al, 2013).

Main actions of metformin

Microarray analyses showed that metformin induces the same gene expression profile as caloric restriction (CR), which was proved to extend life span and health span in various organisms. Metformin acts without any reduction in food intake (Novelle et al, 2016).

Most recently accepted scientific model for the global mechanim of action of metformin

Figure 3 summarizes the cellular mechanism of action of metformin. Outside of the cell (1, top), metformin has the effects on the receptors for cytokines, insulin, IGF-1 and adiponectin. All of them may influence the longevity. Intracellularly (2, middle), metformin inhibits the inflammatory pathway and activates AMPK that inhibits mTOR. The outcome of all these processes (3, bottom) affects inflammation process, cellular survival, stress defense, cell autophagy, and protein synthesis, which have major roles associated with aging (Barzilai et al, 2012). In the following parts of the essay, the action on AMPK, insulin and cytokines will be more described. Other specific modes of action on cancer, mitochondria, vascular endothelium and female reproductive system will be mentioned.

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Action on AMPK in the liver

Metformin treatment results in the phosphorylation and activation of AMP-activated protein kinase (AMPK) in the liver (Gong et al, 2013). AMPK acts on different receptors (Table 3). It’s a major cellular regulator of lipid and glucose metabolism (Figure 3).

Table 3. Receptors of AMPK and their cellular effects in the liver. (Adapted from Gong et al, 2013).

Action linked to anti-hyperglycemia effect

Metformin can reduce blood glucose level by several different mechanisms, especially through non-pancreatic mechanisms without increasing insulin secretion. It increases the effects of insulin; hence, it is termed “insulin sensitizer”. It suppresses endogenous glucose produced by the liver, due to a reduction of gluconeogenesis. It doesn't affect glycogenolysis. In addition, metformin activates the enzyme adenosine monophosphate kinase (AMPK) resulting in the inhibition of key enzymes for gluconeogenesis and glycogen synthesis in the liver while stimulating insulin signaling and glucose transport in muscles. AMPK regulates the cellular and organ metabolism and any decrease in hepatic energy will leads to the activation of AMPK (Figure 3) (Nasri and Rafieian-Kopaei, 2014).

Action linked to anti-oxidant effect

Mitochondria represents as one of the major cellular sources for ROS (Reactive Oxygen Species), with a great number of tissue pathologies, inducing oxidative stress. ROS are implicated in LPS-induced (lipopolysaccharide) IL-1β (proinflammatory cytokine) mRNA production in macrophages (Figure 3). Metformin alters ROS and cytokine production induced by TLR7/8 and TLR9 ligands, thus decreases LPS-induced ROC (Kelly et al, 2015).

Action linked to anti-cancer effect

Backdated analysis of clinical data shows us that inhibition of mTOR (mammalian Target Of Rapamycin) pathway prevents cancer in humans (Blagosklonny, 2008). It is important to know that hyperinsulinemia is also an important factor of cancer development (Anisimov, 2010). Metformin acts as an inhibitor of mTORC1 (mechanistic TOR complex 1) through both AMPK-dependent and independent mechanisms. AMPK activation inhibits the protein kinase mTOR, preventing the phosphorylation of downstream targets, including S6 kinase, ribosomal protein S6, and eIF4E-binding protein 1. Among AMPK-independent mechanisms by which metformin inhibits mTORC1 signaling, it acts to inhibit the Ras-related GTP binding (Rag) GTPases and up-regulate REDD1, a hypoxia-inducible factor 1 (HIF-1) target (Novelle et al, 2016).

Action on mitochondria complex 1

Owen, Doran and Halestrap (2000) showed that 50 mmol metformin inhibited mitochondrial oxidation of glutamate-malate metabolism in hepatoma cells by 13 and 30% after 24 and 60 hours exposure respectively. Succinate oxidation was unaffected. Metformin also leads to time-dependent inhibition of complex 1 in isolated mitochondria, whereas in submitochondrial particles inhibition requires high metformin concentrations (K(0.5),79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1 (Owen, Doran and Halestrap, 2000). Metformin inhibits mitochondrial complex I activity results in cellular respiration decreasement (Novelle et al, 2016) yielding lower ATP levels.

Action on vascular endothelium

The vascular protective actions of metformin are mediated through AMPK activation and subsequent hepatic gluconeogenesis inhibition, fatty acid oxidation as well as an insulin sensitizing action in striated muscle and adipose tissue (Triggle and Ding, 2017).

Action on female reproductive system

Metformin may improve the menstrual regularity of females with polycystic ovary syndrome leading to spontaneous ovulation, and by enhancing the induction of ovulation with clomiphene citrate (Anisimov, 2010).

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Pharmacovigilance of Metformin

Side-effects of Metformin therapy

Gastro-intestinal symptoms

Acute and chronic administration of metformin cause gastro-intestinal symptoms and digestive troubles like nausea and diarrhea . It was reported such common manifestations in differents studies (Dunn and Peter, 1995 ; Jacob et al, 2018, Liu et al, 2014 ; Vecchio et al, 2014). They were estimated to occur in 20% of patients (McCreight et al, 2018; Howlett and Bayley, 1999). Liu et al (2014) reported that diarrhea appeared in 11,3 % of the metformin treated patients. Diarrhea is the most significative metformin-induced side-effects.

In most of the cases, digestive disorders are reversible. Dunn and Peter (1995) estimated a level of non-curable intolerance inferior to 5%. To reduce and eliminate them, metformin doses can be reduced (Hermann, 1979). Moreover, it is possible to avoid them if metformin is absorbed with or after food (Dunn and Peter, 1995; Hermann, 1979).The mechanism of apparition of these disorders is still debated. It may be attributed to local factors in the gut affecting the individual microbiome or intestinal receptors. Further studies are needed (McCreight et al, 2018). In addition, this common digestive incomfort could induce maladsorbtion of nutrients and vitamin deficiency.

Chronic vitamin B12 deficiency

In case of long term use of metformin, cases of anemia and vitamin B12 deficiency have been described and significantly linked to the use of this drug (Hermann,1979). Metformin intake has induced a significant decrease of Vitamin B12 plasma level after 6 weeks to 208 weeks of intake ( Liu et al, 2014). Metformin Dose >2000mg/day has a greater impact on the Vitamine B12 plasma level reduction. Vitamin B12 is a vital nutrient especially for the nervous system (Liu et al, 2014) and cardiovascular system. It acts on the metabolism of a cardiovascular cytotoxic amino acid homocystein (an intermediate product of the methionine metabolism). So, metformin intake could trigger an accumulation of homocystein. Then, it could increase the risk of cardiovascular Disease (CVD). Vitamin B12 deficiency can be remedied by supplementation. Moreover, people at risk of cardiovascular disease are not recommended to use metformin (Figure 4) (Esmaeilzadeh, Gholinezhad-Chari and Ghadimi, 2017).

Hypoglycemia

As metformin has anti-hyperglycemia effects and leads to a decrease of blood sugar level, it can cause hypoglycemia or low blood sugar reaction. This condition has some unpleasant symptoms as dizziness or confusion (Jacob, Garrick and Goldberg, 2018).

Compared to the sulfonylurea (other antidiabetic drug), hypoglycemia conditions are rare and less serious with metformin (Howlett and Bayley, 1999). In that case, it generally causes a very slight hypoglycemia or not at all. It only reduces the glycemia to a normal level. This condition can be affected by several risk factors like low uptake of calories, renal and hepatic diseases, hemodialysis and some specific treatments (glimeperid) (Howlett and Bayley, 1999). Most of this conditions appeared in the contraindications of the drug (Figure 4) . So, if the recommandations are followed, it is very unlikely to cause serious problems (Howlett and Bayley, 1999).

Accumulation of acid lactic and fatal acid lactic acidosis

One of the most important and well studied potential side effects of metformin is acid lactic acidosis. Biguanide class of molecules are known to increase the plasma lactic acid level. This condition can lead to acid lactic acidosis (Vecchio et al, 2014).In most of the studies, lactic acid acidosis is considered as a rare outcome. It has an incidence of about 0.03 cases per 1000 patients-years of treatment. However, if it develops, it is fatal in the half of the cases (Howlett and Bayley, 1999). Lactic acidosis can present clinical signs like anorexia, nausea, vomiting and abdominal pain. More severe cases can be marked by hypotension, respiratory failure, arrhythmia and hypothermia (Prikis et al, 2007).

Controversial results about a link between an increase in lactic acid level in the blood and metformin uptake were found (Vecchio et al, 2014). So, a classification of the lactic acidosis types was established based on its level of association with metformin (Vecchio et al, 2014). 4 categories are distinguished from no link to a direct induction: metformin-unrelated lactic acidosis (MULA)‘metformin-induced lactic acidosis (MILA)’ (exclusively due to metformin),‘metformin-associated lactic acidosis (MALA)’ lactic acidosis in metformin therapy (LAMT) (if metformin concentration is not available). There is a complex association between acidosis and metformin admnistration (Lalau et al, 2017). A lot of factors can influence this association. They include most of the factors which are present in the contradictions of that drug (Figure 4).

Risk of ketoacidosis

Metformin treatment may increase blood ketoacid level (Buse et al, 2017). MALKA (Metformin Associated ketoacidosis) was reported to have fatal consequences (Schwetz et al, 2017). The ketogenesis is induced by the mechanism of action of metformin by inhibiting the hepatic gluconeogenesis (Schwetz et al, 2017; Bonsignore et al, 2014). In combination to MALA and renal diseases, other factors can influence the production of ketoacid like pregnancy and alcohol intake (Schwetz et al, 2017). So, those factors are known to be contraindications to use metformin (Figure 4).

Contraindications and Precautions for metformin treatment

Most of the contraindications listed in the prescription can cause the most detrimental side effects. So, they are in majority linked with acid lactic acidosis. Two types of contraindications can be reported : contraindications linked to the health status of the patient and contraindications linked to drug interactions (Figure 4).

Contraindications linked to the health status, medical condition of the patient

As most of metformin is eliminated by the kidney, renal disease or dysfonction is the most important risk factor that can found in the different studies as it has an impact on the clearance of the drug (Lalau et al, 2017). Renal function anomaly is generally detected when serum creatinine level > 130 micromol/L or > 1.5 g/L) . In that case, most of the metformin can't be eliminated. So, it tends to accumulate and causes the adverse effects. Hepatic disease is another key factor in the production of lactic acid (Lalau et al, 2017). As metformin increases the uptake of glucose by the liver, the liver converts extra glucose to extra lactic acid and sends it in the plasma. If the liver has impaired functions lactic acid isn't removed and is accumulated in the plasma. So, Howlett and Bayley (1999) showed that the most serious side effects occur if the patients are wrongly prescribed the drug. It only happens if the recommandations and contraindications are not respected. Other contradictions like congestive heart failure which are in link with hypoxic states have to mentioned. Some characteristics of the patient (age and pregnancy) should be checked as well. This conditions have an influence on the normal function of the kidney and may influence the acid lactic accumulation (Figure 4) (Lalau et al, 2017).

Contraindications due to Drug to Drug interactions

One of the most well-known drug interactions to metformin is the use of ionidated contrast media. That kind of treatment increases the risk of lactic acidosis (Figure 4). Medications that deteriorate renal functions like angiotensin-converting enzyme inhibitors ACEi, angiotensin receptor blockers, ARB and non-steroidal anti-inflammatory drugs NSAI have to be avoided (Figure 4) (Visconti et al, 2016). Other drug like carbose ; cephalexine cimetidine ; dolutegravir ; pyramethamine, ranolazine; trimethoprim and tyrosine kinase inhibitors treatment are included in the exclusion factors of some studies (McCreight et al, 2018). They are known to influence the pharmacokinetics of metformin. Drug to Drug interactions between metformin and other drugs are still discussed. Individual factors and pharmacogenetics of metformin require further studies (Visconti et al, 2016) .

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Conclusion

• In most of studies, there is a significant association between metformin therapy and anti-aging effects. It reduces or delays the age-related diseases like most of the fatal cancers. So, it may significantly increase the lifespan and the living conditions of older people. Concerning the laboratory studies on animals, results are controversial. Individual factors, age, sex, species and strains, genetic factors, mode of consumption of metformin (duration, dosage, age when to start the therapy) may have a significant impact on the results . Only, studies on female mice of specific strains significantly showed an increase in lifespan and a reduction in age-related cancers. Metformin is an oral drug which is not metabolized and is excreted unchanged in the urine. Its mechanism of action is not totally understood. Metformin mainly acts on the body by stimulating a membrane or intracellular receptor called AMPK . Then, AMPK activates or inhibits other receptors and pathways in different parts of the body. Their final effects is associated to cellular wellbeing and longevity. Metformin therapy may cause several and a great variety of positive effects and advantages. Nevertheless, some adverse effects were reported. Gastro-intestinal problems are the most frequent disorders but they are reversible and not too severe. Individual risk factors like principally hepatic or renal diseases can influence the occurence of side effects. Metformin intake was proved to be in relation or to cause fatal consequences. Acid Lactic acidosis is the most significant fatal outcome. Nevertheless, if metformin is taken while respecting the recommendation of use, it is very unlikely to cause any serious and irreversible problems. Other individual pharmacogenetic factors require further studies to explain the heterogenous inter-individual answer to metformin. Compared to other anti-hyperglycemic drug like other common biguanides (phenformin), the side effects are relatively minimal. So, the benefit/risk ratio is significantly superior. Metformin can be considered as a safe drug (Hermann, 1979). New studies suggest that metformin could have a positive effect on other systems and tissues like the skin, cognitive functions or even the mood. Thus, all the secrets of metformin and fields of application are far from being completely known and understood.

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