Targeting multiple hallmarks of mammalian aging with combinations of interventions

Combination therapy in mammals

Table 2 summarizes studies that examined the effects of combination therapies including both genetic and pharmaceutical interventions on normally aging mammals and led to additive or synergistic increases in lifespan. Table 3 contains known negative results of combination therapies. Table 4 provides examples of combination therapies studied on therapies tested on progeroid and disease mouse models. Progeroid animals are often used as a model for aging because of convenient short initial lifespan, but do not necessarily represent the normal aging process and were thus analyzed separately. Table 5 contains studies of combination therapies that showed various health benefits, but in which lifespan extension was not studied.

Table 2. Combined therapies aimed at different aging hallmarks, most effective in rodents.

Therapy	Reasoning	Target HA	HA over lap	Biological effects	Effect of combination	Separate interventions, median lifespan compared to control	Combined interventions, median lifespan compared to control	Separate interventions, maximum lifespan compared to control	Combined interventions, maximum lifespan compared to control	Ref.
Metformin + rapamycin: 1000 ppm and 14 ppm in food from 9 months of age (ITP study)	Both components inhibit different mTOR complexes, and metformin is expected to compensate negative effects of rapamycin	DM, LP, DNS	overlap	Increase in lifespan	Synergistic, except female maximum;	metformin: 0% ♀ + 7% ♂ rapamycin: + 21% ♀ + 13% ♂	+ 23%	metformin: 0% ♀ - 2% ♂ rapamycin: + 11% ♀ + 8% ♂	+ 17% ♀ + 10% ♂	[47] [44]
Simvastatin + ramipril: 20 and 5 mg drug/kg bw/d in feed	Combination of drugs effective in decreasing mortality and is used in treatment of aging-related pathologies in humans	DNS		Decreased weight, higher serum glucose levels, increased number of benign tumors and diathesis, which did not affect the lifespan	Synergistic	Simvastatin: 0%* Ramipril: + 3%*	+ 9% mean and median	Simvastatin: - 4.1%* Ramipril: + 2%*	+ 5%*	[89]
Rapamycin + acarbose started at 9 m.o.: 14.7 ppm and 1000 ppm in feed (ITP study)	Components target different but overlapping pathways. Rapamycin inhibits mTOR, acarbose reduces glucose levels	DM, LP, DNS, AMP	overlap	Mice gained less weight than controls	Single-component treatment n.a. in this study, comparing with other studies additive in males, neutral in females	n.a.	+ 28% ♀ + 37% ♂	n.a.	+ 21% ♀ + 24% ♂	[90]
Rapamycin + acarbose stated at 16 m.o.: 14.7 ppm and 1000 ppm in feed (ITP study)							+ 12% ♀ + 14% ♂		+ 15% ♀ + 18% ♂
Glycine + N-Acetylcysteine (GlyNAC): 1.6 mg/g feed and 1.6 mg/g feed each compound	GlyNAC was shown to improve aging-related dysregulations of multiple biological processes	MitD, GI, DNS, DM	partial overlap	In old mice glutathione levels, markers of oxidative stress, mitochondrial dysfunction, autophagy and genomic damage returned to youthful levels	Synergistic, based on other studies with separated components	n.a.	+ 24%	n.a.	+ 33%*	[91]
Dasatinib + quercetin: started at 24 m.o., 5 mg/kg and 50 mg/kg; biweekly orally for three days	Targeting senescent cells late in life	CS, Inf	overlap	Senescent cell clearance, improved physical activity	n. a.	n.a.	+ 6%; + 36% median post-treatment lifespan	n.a.	+ 5%	[92]
p53/TERT + p16/Arf (transgenic mice)	Tumor-suppressing transgenes to compensate tumor-inducing effect of TERT	GI, TA	Partial overlap	Decreased tumor formation, improved physical function, intestinal barrier function, glucose tolerance and genome integrity, reduced telomere	Synergistic	p53/TERT: n.s. p16/Arf: + 7%	+ 40%	p53/TERT: + 7% p16 + Arf: n.a. in this study	+ 22%*	[13]
p53 + s-ARF (+p15/p16)	Combination of oncosupressors	GI	overlap	Decreased tumor formation, improved genome stability, lifespan extension	Synergistic	p53: n.s. s-Arf: + 7%	+ 16%	p53: n.s. s-Arf: + 5%	+ 10%*	[93]
Catalase OE in peroxisomes + superoxide dismutase overexpression (in transgenic mice)	To decrease reactive oxygen species levels	MitD		Lifespan extension	Synergistic	SOD1: n.s. PCAT: + 13%	+ 19%	SOD1: n.s. PCAT: + 6%	0%	[94]
OCT4 + SOX2 + KLF4 (via viral vectors. Induction of OSK started at 124 weeks old mice)	Epigenetic reprogramming	SCE, EA	overlap	Decreased frailty index and extended lifespan	n.a.	n.a.	+ 109% median post-treatment	n.a.	+ 11%*	[95]
Ames mice (PROP1 deficient) + CR	Targeting insulin sensitivity	AIC, DNS, DM, LP	overlap	Lifespan extension	Additive	Ames: + 38% CR: + 27%	+ 69%	Ames: + 39% CR: + 35%	+ 61%	[96]
GHRH KO + CR	Targeting insulin sensitivity	AIC, DNS, DM LP	overlap	Lifespan extension	Additive	KO: + 45% CR: + 21%	+ 64%*	KO: + 30% CR: + 23%	+ 52%*	[97]
Ames mice (PROP1 deficient) + CR	Targeting insulin senstivity	AIC, DNS, DM, LP	overlap	Further increased insulin sensitivity	Additive	Ames: + 36%* CR: + 26%*	+ 68%*	Ames: + 39%* CR: + 35%*	+ 61%*	[98]
GHR KO + CR	GH/IGF-1 axis suppression	AIC, DNS, DM, LP	overlap	Lower weight	Additive for female’s maximum lifespan	KO: + 31% CR: + 25%	+ 31%	KO: + 17% CR: + 17%	+ 26%	[99]
Suppressed GH + calorie restriction (transgenic rats)	GH/IGF-1 axis suppression	AIC, DNS, DM, LP	overlap	Lower weight, protected rats from age-related kidney pathologies	Additive	Suppressed GH: + 7% CR: + 11%*	+ 25%*	Suppressed GH: + 10% CR: + 18%	+ 30%	[100]
Abbreviations: GI: genomic instability; TA: telomere attrition; EA: epigenetic alterations; LP: loss of proteostasis; DNS: deregulated nutrient-sensing; Mitd: mitochondrial dysfunction; CS: cellular senescence; SCE: stem cell exhaustion; AIC: altered intercellular communication; DM: disabled macroautophagy; Inf: inflammation; IA: immunaging; AMP: Altered mechanical properties. Lifespan extension values deduced from provided mortality curves are marked with (*). N.A. is not applicable; n.s. is not statistically significant (mentioned as in article).

Table 3. Combined therapies aimed at different aging hallmarks with no additive or synergistic effect on mammalian lifespan.

Therapy	Reasoning	Targeting HA	Biological effects	Effect of combination	Median lifespan compared to control	Maximum lifespan compared to control	Ref.
PROP1 deficiency + GHR KO	Combining well-known dwarf mutations that extend lifespan	DNS overlap	Improved glucose and insulin tolerance	Lifespan did not exceed the effect of separate interventions	+ 52% ♀ + 30 % ♂	+ 53% ♀ + 25 % ♂	[129]
CuZnSOD + MnSOD (antioxidant enzymes)	Suppression of oxidative stress	n. a.	n. a.	No effect	0%	0%	[124]
Pycnogenol + quercetin + taxifolin	Suppression of oxidative stress	MitD, Inf	Didn’t extend mice lifespan	N.A.	n.s.	n.s.	[128]
Green tea extract + black tea extract + morin	Suppression of oxidative stress	MitD, Inf	−	N.A.	n.s.	n.s.	[128]
GHR KO + intermittent fasting	Metabolism	DNS, AIC	n.a.	No effect	n.s.	n.s.	[117]
GHR KO + 30% CR (from 2 month)	Metabolism	DNS, AIC	n.a.	No effect	n.s.	n.s.	[99]
GHR KO + CR	Metabolism	DNS, AIC	n.a.	Lifespan did not exceed the effect of separate interventions	n.s.	n.s.	[98]
Abbreviations: GI: genomic instability; TA: telomere attrition; EA: epigenetic alterations; LP: loss of proteostasis; DNS: deregulated nutrient-sensing; MitD: mitochondrial dysfunction; CS: cellular senescence; SCE: stem cell exhaustion; AIC: altered intercellular communication; DM: disabled macroautophagy; Inf: inflammation; IA: immunaging. Lifespan extension values estimated from mortality curves are marked with (*). N.A. is not applicable; n.s. is not statistically significant (mentioned as in article).

Table 4. Combined therapies aimed at different aging hallmarks, effective in progeroid or disease mammal models.

Therapy	Targeting HA	Biological effects	Effect of combination	Median lifespan compared to control	Maximum lifespan compared to control	Ref.
Pravastatin + zoledronate	LP	Reduced progeroid symptoms and extended the lifespan	Synergistic	+ 77%	+ 80%	[136]
ATP + levamisole (TNAP inhibitor) + ARL67156 (eNTPD inhibitor)	AMP	Extended lifespan and improved symptoms in progeria animals by preventing vascular calcification	Synergistic	+ 12%	+ 13%	[134]
CR + fish oil	DNS	Reduced inflammation and increased lifespan	Additive	+ 166%	+ 167%	[138]
Cyclophosphamide + tilorone		Increased lifespan but very slightly compared with cyclophosphamide alone	Additive	+ 73%	N.A.	[139]
OSKM	SC, EA	Reduced progeroid symptoms and extended the lifespan	Synergistic	+ 20%	+ 21%	[137]
Abbreviations: GI: genomic instability; TA: telomere attrition; EA: epigenetic alterations; LP: loss of proteostasis; DNS: deregulated nutrient-sensing; MitD: mitochondrial dysfunction; CS: cellular senescence; SCE: stem cell exhaustion; AIC: altered intercellular communication; DM: disabled macroautophagy; Inf: inflammation; IA: immunaging. N.A. is not applicable.

Table 5. Combined therapies aimed at different aging hallmarks without data on lifespan extension.

Therapy	Reasoning	Targeting HA	Biological effects	Effect of combination	Ref.
FGF21 + αKlotho + sTGFβR2	Chosen genes play roles in aging-associated diseases and involved in HA processes, they are involved in separated pathways	AMP, DNS, AIC, IA	Some combinations rescued mice from modeled aging-associated diseases	Synergistic for TGFβ+ FGF21 and TGFβ + αaKlotho; Negative for FGF21 + αaKlotho	[82]
Ezh2 expression + JmjD3 knockdown in beta cells of pancreatic islets	EZH2 is decreased in aging b-cells, but it’s OE alone wasn't enough to repress ink4a, which was achieved by adding KD of Trx component JmjD3	EA	Improved beta cell replication	Synergistic in aged mice	[130]
Oct4, Sox2, Klf4 and c-Myc, long-term partial induction	Induction of stemness	SCE, EA	Rejuvenation of tissues and whole organism		[121]
NK-cells infusion + dopamine	Removing senescent cells	CS	Dopamine enhanced senolytic activity of NK cells	Synergistic	[112]
Dasatinib + quercetin	Removing senescent cells	CS	Improve health in aged mice, extends healthspan in progeroid mice	Additive, targets more types of senescent cells than each drug alone	[12]
Resveratrol + copper	Elimination of the extracellular chromatin particles	TA, MitD, CS, GI, Inf	Reduced aging-associated pathologies in the cells of aging brain	Synergistic	[126]
Rapamycin + acarbose + phenylbutyrate	All components show anti-aging effects and target different but overlapping pathways. Rapamycin inhibits mTOR, acarbose reduce glucose levels, phenylbutyrate improved mice cognitive and physical condition	DNS, Inf, CS, AMP	Delays aging phenotypes, improves healthspan, cognitive and physical condition, reduces tumor burden	Synergistic	[86]
Dasatinib + Quercetin: a single 3-day oral treatment regimen with D 100 mg daily and Q 1000 mg total daily (500 mg twice daily)	Removing senescent cells	CS	Decreased senescent cell burden in adipose tissue and epidermis in human with diabetic kidney inflammation	N.A.	[108]
Growth hormone + metformin + DHEA (experiment performed on human patients)	rhGH was used based on prior evidence that growth hormone (GH) has thymotrophic and immune reconstituting effects in animals. Because GH might induce hyperinsulinemia which is an undesirable side effect, dehydroepiandrosterone (DHEA) and metformin were added in an attempt to limit the “diabetogenic” effect of GH	EA, DM, LP, DNS	Regenerative response in thymus and bone marrow, delayed epigenetic clock	Synergistic	[131]
Intra-ocular AAV-delivered Yamanaka factors (OSK)	Known effect of epigenetic clock reversal	EA	Regeneration of damaged optic nerve, recovery of vision in mice with glaucoma, epigenetic clock reversal	Synergistic	[132]
Metformin and Glycyrrhiza uralensis Fischer extract	Known anti-obesity agents	DM, LP, DNS	Prevented hepatic steatosis and adiposity, improved glucose homeostasis and reduced inflammation in mice on high-fat diet	Synergistic	[140]
Dasatinib + quercetin	Removing senescent cells	CS	Attenuate adipose tissue inflammation, and ameliorate metabolic function (mice)	Synergistic	[141]
Dasatinib + quercetin	Removing senescent cells	CS	Ameliorates age-dependent intervertebral disc degeneration in mice	Synergistic	[104]
Dasatinib + quercetin	Removing senescent cells	CS	Prevents uterine age-related dysfunction and fibrosis in mice	Synergistic	[106]
Dasatinib + quercetin: 100 mg/day + 1250 mg/day; three-days/week over three-weeks orally	Removing senescent cells	CS	Improved physical performance in patients with idiopathic pulmonary fibrosis (IPF)	Synergistic	[109]
GlyNAC: glycine (1.33 mmol/kg/day) and N-acetylcysteine (0.81 mmol/kg/day), every 4-weeks for 24-weeks	Metabolism	DNS	GlyNAC supplementation in older adults improves glutathione deficiency, oxidative stress, mitochondrial dysfunction, inflammation, insulin resistance, endothelial dysfunction, genotoxicity, muscle strength, and cognition: Results of a pilot clinical trial	Synergistic	[142]
Abbreviations: GI: genomic instability; TA: telomere attrition; EA: epigenetic alterations; LP: loss of proteostasis; DNS: deregulated nutrient-sensing; MitD: mitochondrial dysfunction; CS: cellular senescence; SCE: stem cell exhaustion; AIC: altered intercellular communication; DM: disabled macroautophagy; Inf: inflammation; IA: immunaging. N.A. is not applicable.

Combinations that target different hallmarks of aging

The majority of combination therapy studies in mammals focused on complementing therapies that targeted the same hallmark or set of hallmarks of aging (see Table 2). There are only a few exceptions. First, the combination of TP53, p16 and TERT enhancing mutations demonstrated a strong synergistic effect on the lifespan extension [13]. Initial targeting of only two HAs (GI and TA) made mice more resistant to cancer and telomere loss, improved their physical condition, and slowed down the development of aging-associated pathologies and frailty.

Second, a combination gene therapy consisting of three longevity-associated AAV-delivered genes, namely fibroblast growth factor 21 (FGF21), αKlotho and a soluble form of mouse transforming growth factor-β receptor 2 (sTGFβR2) was shown to be beneficial on several mouse models of age-related diseases, such as obesity, type II diabetes, heart failure, and renal failure. Interestingly, the therapy action was synergistic for TGFβ+FGF21 and TGFβ+αKlotho, while the effect was negative for FGF21+αKlotho (the combination worked worse than the individual intervention results for all 4 diseases) [82]. The components of the therapy target different signaling pathways and can be related to the AMP and AIC hallmarks. The triple combination did not outperform either of the two best double combinations by its effects on age-related pathologies. Unfortunately, the effects of these therapies on lifespan were not reported [82].

Combinations based on rapamycin

Among the pharmacological combination therapies that targeted the same HAs many involved the mTOR inhibitor rapamycin in conjunction with other molecules that affect the DNS hallmark, such as, the antidiabetic inhibitor of liver glucose production metformin, and inhibitor of α-glucosidase acarbose. While metformin and acarbose are readily used as anti-diabetic drugs, chronic rapamycin treatment has been shown to increase glucose intolerance in mice [83, 84]. This is one potential explanation for the increased benefit of the combination. Although some researchers argue that the diabetes-like condition caused by high doses of rapamycin might actually be beneficial in terms of aging by itself.

Combinations of rapamycin and metformin have resulted in an additive effect on mouse lifespan [47], while combinations of rapamycin and acarbose have led to an additive increase in male lifespan with no additive effect in females [85] (if compared to studies where rapamycin [44] or acarbose [75] were used as a monotherapy). One additional study tested a combination of rapamycin, acarbose and phenylbutyrate [86] (a drug that was previously shown to rescue cognitive impairment in a mouse model for Alzheimer diseases) [87]. The combination worked better than individual treatments in decreasing tumor burden as well as cognitive and physical decline in aged mice. Performance tests favored the combination and rapamycin alone comparing to the other two drugs. All drugs reduced the severity of lesions, but the best results were observed for the combination therapy.

Rapamycin and metformin have some overlapping targets and mechanisms of action: mTOR pathway inhibition, enhancing insulin/IGF1 signaling, and autophagy activation. Acarbose affects glucose metabolism by slowing down breakage of polysaccharides to glucose in the intestine. Phenylbutyrate promotes the catabolism of branched-chain amino acids and correspondingly downregulates mTOR [88].

Combinations of drugs used against cardiovascular disease

A combination of the statin simvastatin and the angiotensin-converting enzyme inhibitor ramipril cause a small yet synergistic increase of male mouse lifespan [89]. Simvastatin lowers low-density lipoprotein (LDL) via reversible inhibition of HMG CoA reductase. Neither of the drugs was shown to increase normal rodent lifespan [43, 101], but they did so in combination. While the mechanisms of action of these drugs are different, they both target similar HAs, namely altered intercellular communication (AIC). One important side-effect of the statin and combined treatment was the increased occurrence of benign lung tumors and hemorrhagic diathesis, but this did not affect overall survival according to the study [89].

It should be noted, that translating any of these findings to humans is particularly difficult because unlike humans mice rarely develop coronary artery atherosclerosis with age, do not exhibit the same high density lipoprotein (HDL) profiles, and have other important difference that affect their cardiovascular system [102].

Combinations that target senescent cells

The use of senolytics dasatinib + quercetin (D+Q) is another example of the approach where both interventions target the same HA – cellular senescence. Quercetin alone does not improve murine lifespan [103]. The life-extending properties of dasatinib alone have not been investigated, but it was shown that its combination with quercetin is more efficient in elimination of senescent cells and prolongs the lifespan of mice. The oral administration of dasatinib and quercetin decreased the number of naturally occurring senescent cells and their secretion of frailty-associated proinflammatory cytokines in explants of human adipose tissues. Moreover, in both senescent cell–transplanted young mice and naturally aged mice the combination therapy alleviated physical dysfunction and increased post-treatment survival by 36% [104]. Other trials have shown that dasatinib plus quercetin can improve metabolic function, attenuate adipose tissue inflammation and age-dependent intervertebral disc degeneration [105] as well as prevent age-related uterine dysfunction and fibrosis [106].

Dasatinib is a senolytic agent, inhibiting tyrosine kinases, while the antioxidative flavonoid quercetin apart from senolytic activity via inhibition the anti-apoptotic protein Bcl-xL is anti-inflammatory [107]. Additionally, the combination of senolytics dasatinib and quercetin was shown to selectively remove senescent cells in a phase 1 study on nine human patients suffering from diabetic kidney disease [108]. Another study on 14 patients with idiopathic pulmonary fibrosis (IPF) showed that intermittent administration of D+Q slightly but significantly improved their physical functions such as grip strength [109]. In patients with diabetic kidney disease D+Q treatment decreased senescent cell burden in adipose tissue and epidermis [108]. In both human trials a so-called “hit-and-run” administration of senolytics was applied, which is either single dose or intermittent administration. This is rationalized by several reasons: first, senescent phenotypes develop slowly, thus after elimination of senescent cells new ones will not appear soon. Second, the senolytics’ mechanism of action does not require them to be continuously present. Finally, such a regimen was shown to reduce the side effects of dasatinib [110, 111].

Another promising approach to remove senescent cells was demonstrated on a mouse model by applying adoptive infusion of senolytic natural killer (NK) cells boosted by acein, a dopamine-releasing peptide, which caused no senolytic effect by itself. The therapy resulted in reduced local and systemic senescence-associated secretory phenotype (SASP) in aged mice and enhanced the elimination of senescent cells compared to NK cell infusion alone [112].

Combinations based on calorie restriction

Calorie restriction (CR) is one of the most studied methods of life extension in mice, rats and other mammals [113]. The mechanism of lifespan extension through CR is usually attributed to a number of pathways that involve IGF1/FOXO [114], mTOR [115], SIRT1 [116] and other actors. Mice with genetically determined dwarfism (GH, GHR, GHRHR, PROP1 mutations that suppress the GH/IGF1 axis) are well known for their increased lifespan, and there is a number of studies in which such mice were fed with calorie-restricted diets. In most cases such combinations extended the animal’s lifespan even further. For example, PROP1 deficiency alone extended lifespan by 37%, and in combination with CR by up to 69% compared with wild-type ad libitum fed controls [96]. The potential mechanism of lifespan extension in mice by PROP1 deficiency and by CR could be mediated by increased insulin sensitivity, which is further improved by this combination [98].

In rats suppression of GH also extended lifespan, and calorie restriction enhanced this effect [100]. This effect appears to be additive rather than synergistic. The main exceptions are GHR knockout mice that are less responsive to diet restrictions. CR failed to increase overall, median, or average lifespan in these mice and increased maximum lifespan only in females and to a rather small extent (9%) [117]. Also, the combination of GHR KO and CR does not improve insulin sensitivity more than the separate interventions [98].

Combinations based on partial epigenetic reprogramming

The genes Oct4, Sox2, Klf4 and c-Myc are the four Yamanaka factors (OSKM) that can be used in combination to reprogram specialized cells such as fibroblasts into induced pluripotent cells [118, 119]. Short-term activation of these genes can be used for partial epigenetic reprogramming that does not lead to complete loss of cellular identity but reduces the biological age of targeted cells as measured by epigenetic clocks and other markers of aging [120]. We can classify each OSKM component as aiming for the EA and SCE hallmarks of aging, thus the targets of this therapy overlap.

Virus delivery of the OSK subset of Yamanaka factors to extremely old mice (124 weeks old) decreased their biological age measured by methylation epigenetic clocks, improved their health (decreased frailty) and extended median lifespan by 6% [95]. Another study, which utilized a full set of Yamanaka factors, showed that their long-term partial induction led to the reversion of the epigenetic clock and reduced the expression of numerous genes associated with inflammation, senescence, and stress response, resulting in improved tissue rejuvenation. The phenotype changes were detectable in different tissues, such as the kidney and skin. Epigenetic clock reversal as well as metabolic and transcriptomic changes, including downregulation of pro-inflammatory genes and cellular senescence could be observed after treatment, with the degree of manifestation dependent on the duration of OSKM expression [121].

Combinations of tumor suppressors

Synergistic lifespan extension can be achieved not just by targeting the same HAs but the same biological processes, e.g., elevated expression of oncosupressors p53 and Arf extended mice median lifespan 16%, while p53 alone only decreased tumor incidence, and Arf had a very weak effect on lifespan [93]. The addition of a TERT transgene extended the lifespan of p53/Arf over-expressing mice by 40%. While p53 and Arf alone protects mice from cancer, neuromuscular degeneration and oxidative damage, addition of TERT attenuated the accumulation of histone markers of double-strand breaks, particularly at telomeric regions, and reduced inflammation [13].

Combinations that target oxidative stress

According to the free-radical theory of aging, lifespan can be increased by exogenous administration of antioxidants or molecules that enhance the organism’s ability to remove notable oxidants such as H₂O₂ [122]. This theory has a number of problems, including observations that certain antioxidants including vitamins A and E may actually increase mortality [123]. One possible explanation is that the introduction of additional antioxidants may negatively affect the endogenous antioxidant systems. Nevertheless, a number of studies tested combinations of interventions aimed at reducing oxidative stress.

One such intervention targeted a single HA (MitD) via overexpression of peroxisomal catalase and superoxide dismutase (PCAT and SOD1 genes) had the synergistic effect on the median but not maximal lifespan [94]. Another study examined the effects of Cu and Zn superoxide dismutase overexpression separately or together, but none of the interventions extended the lifespan of mice [124], contrasting the results of similar experiments on Drosophila [125].

Antioxidants are often used to combat aging-related diseases, but there are examples where pro-oxidants provide similar effects. A pro-oxidant combination of chelated copper and resveratrol creates free oxygen radicals which eliminate cell-free chromatin particles originating from dead cell debris that can cause inflammation. Thus, in this case pro-oxidants are anti-inflammatory. Application of the treatment to aged mice led to a reduction of several hallmarks of aging in brain cells including TA, GI, Inf, CS and MitD. The therapy also resulted in significant downregulation of glucose blood levels, cholesterol, amyloid deposition, and C-reactive protein [126].

Another study reported that a combination therapy of glycine and N-acetylcysteine (Gly-NAC) given together significantly prolonged the median and maximum lifespan of mice by 23.7% and 33.1% respectively [91]. Both compounds are claimed to act through ameliorating the age-associated glutathione deficiency and reducing oxidative stress. Glycine itself was previously shown to have only a very modest (4–6%) effect on lifespan [81] while N-acetylcysteine had no effect on female lifespan, but provided a 44% increase in median lifespan in males [127]. We noticed that in the N-acetylcysteine study male mortality appears to be unusually high in the control group, so we doubt that single N-acetylcysteine provided lifespan increase.

Various natural products with presumed antioxidant and anti-inflammatory properties are considered as potential geroprotectors, however many of them do not show any pronounced effects on murine lifespan. The combination of pycnogenol, quercetin and taxifolin, did not expand lifespan of the experimental animals. The same conclusion was made for single treatments of blueberry, pomegranate, green and black tea, cinnamon, sesame, French maritime pine bark, green tea extract, black tea extract and morin. The authors suggest a number of previous reports that these natural products expanding longevity may have resulted from induced caloric restriction [128].

Combinations that were studied for anti-aging effects other than lifespan extension

Some studies of combination therapies were designed to test if they can reverse separate aging-related phenotypes and/or pathologies without attempting to register any effect on general lifespan.

One combination gene therapy against type II diabetes was aimed at overexpressing the transgenic EZH2 protein, a key component of the Polycomb complex, while simultaneously knocking down the MII1 gene, a part of the Trithorax complex. It resulted in successful repression of the Ink4a locus and rejuvenation of the pancreatic islets through promotion of β-cell proliferation. Notably, the EZH2 alone was not sufficient to cause improvements in mice older than 8 months while simultaneous MII1 knockdown acted well even in aged animals. Additionally, a combination of EZH2 overexpression and JnjD3 knockdown in beta cells of aged mice enhanced replication of beta cells, unlike the separate interventions [130].

There is also limited evidence that some combination therapies can ameliorate age-related phenotypes in humans. For example, a combination of growth hormone (GH), dehydroepiandrosterone (DHEA) and metformin was used on a group of 10 patients aged 51–65 years. GH was aimed at thymus regeneration and reduction of immunosenescence, while two other drugs were supposed to reduce the diabetogenic side effects. Indeed, the therapy resulted in regenerative response in thymus and bone marrow and delayed epigenetic clock advancement in the studied tissues [131].

A prominent study used a combination of three Yamanaka factors (excluding the carcinogenic c-Myc) to regenerate damaged optic nerves and recover vision in mice with glaucoma served epigenetic age reversal. No pronounced side effects were observed [132].

Effect of combination therapies on lifespan of progeroid and disease animal models

Although our review focuses mainly on experiments where therapeutic interventions were able to significantly expand the lifespan of normally aging lab mice, we decided to make a separate overview of combination therapies which prolonged lifespan and healthspan in progeroid and other rapidly aging mouse models. Despite progeroid models being less representative of the normal aging processes, studies on them still remain an important source of knowledge about the mechanisms of aging and anti-aging treatments [133].

In a well-known Hutchinson-Gilford Zmpste24 −/− progeria mouse model of premature aging, a combination of three synergistically acting drugs targeting the AMP hallmark (by preventing vascular calcification) has shown a significant though moderate median lifespan increase. The application of extracellular ATP yielding pyrophosphate (which is a known inhibitor of calcium-phosphate deposition and therefore of vascular calcification [134]) was boosted by two drugs: tissue-nonspecific alkaline phosphatase (TNAP) inhibitor levamisole and ectonucleoside triphosphate diphosphohydrolase (eNTPD) inhibitor ARL67156. The drugs enhanced the production of pyrophosphate from ATP and reduced its hydrolysis, respectively, leading to prevention of vascular calcification. Notably, ATP alone did not play any effect on the lifespan of studied animals [135].

A combined treatment with statins (pravastatin) and aminobisphosphonates (zolendronate) was enough to synergistically extend longevity in the same mouse model. Both drugs target the same hallmark of aging, namely the loss of proteostasis, by simultaneously inhibiting two interchangeable pathways of prelamin A and progerin prenylation. The therapy significantly prolonged median and maximum lifespan (Table 4) and evidently improved the aging-like phenotypes typical for the model, including growth retardation, loss of weight, lipodystrophy, hair loss and accumulation of bone defects [136].

Another progeroid mouse model was efficiently treated by the combination of Yamanaka factors (OSKM). LAKI mice carrying a truncating lamin mutation were subjected to short-term cyclic OSKM induction, which resulted in amelioration of cellular and physiological hallmarks of aging and led to an extension in median and maximal (up to 20%) lifespan [137].

Autoimmunity is a well-known age-dependent pathology. Studies of autoimmune responses often involve the autoimmunity prone NZB × NZW F1 mice model. On this model the additive effect of calorie restriction and fish oil, targeting the DNS and INF hallmarks was observed. The combination was shown to reduce levels of several important proinflammatory factors, including TNF-α and NF-kB and to upregulate antioxidant pathways leading to a dramatic median lifespan increase of 166% [138]. Another experiment on the same model involved mice treatment with a known immunosuppressant cyclophosphamide along with an immunostimulator tilorone. This combination of drugs exhibited an additive effect on lifespan extension [139].

Developing combination therapies to enhance mammalian longevity

It appears that combination therapy is a promising approach for mammalian lifespan extension. It can work when targeted hallmarks of aging are different and even in cases when the presumed hallmarks overlap. However, the number of tested combinations of longevity-enhancing treatments is relatively small and most studies are done in rodents. The design and implementation of additional studies is warranted, including those in other species of mammals.

The current availability and diversity of lifespan promoting therapies allows the possibility to design studies in mice targeting at least thirteen hallmarks of aging simultaneously. Nine of these hallmarks can be readily targeted in animals of any genetic background. For example, this can be accomplished with a combination of dasatinib plus quercetin, FGF-21, acarbose, methionine restriction, young bone marrow transplantation and gene therapy for VEGF and TERT activation and hypothalamus-restricted Nf-Kb inactivation (example combination 1, Figure 1). Three more hallmarks can be targeted by genetically engineering mice overexpressing SIRT6 and by creating mclk1 knockout heterozygotes (example combination 2, Figure 1). Theoretically the same effects can be produced by experimental gene therapies. However, example combinations 1 and 2 involve surgical procedures that by themselves can be traumatizing, a lentiviral gene therapy that can cause insertional mutagenesis, a CMV gene therapy that in theory may contribute to immune system depletion, an AAV gene therapy that like the other two gene therapies may cause immune reactions and liver toxicity.

Figure 1. Possible strategies of development of aging combination therapies. This figure illustrates the various hallmarks of aging that could be addressed by different existing interventions. Additionally, it proposes four example combinations of therapies, each detailing which specific treatment targets a particular hallmark of aging.

Recently the LEVF foundation initiated an experiment to test a combined therapy of rapamycin, a senolytic called navitoclax, hematopoietic stem cell transplantation (HSCT), and telomerase expression (example combination 3, Figure 1) [143]. This approach would target six hallmarks of aging: deregulated nutrient sensing (DNS), disabled macroautophagy (DM), loss of proteostasis (LP), cellular senescence (CS), stem cell exhaustion (SCE) and telomere attrition (TA).

Given that there are many possible interventions that have provided lifespan extension in mice as single therapies, for the conservation of effort and resources, it would be reasonable to develop strategies for prioritizing the most promising combined therapies. Ideally, these strategies should take into account the cost and effectiveness of individual treatments, their theoretical interactions and whether the targeted hallmarks overlap. We would also argue that we should prioritize interventions that can be applied to already aged animals given the ultimate practical goal of prolonging the lifespan of existing humans. Perhaps interventions that are evidently less effective in humans than in mice, dangerous or difficult to sustain should be deprioritized for practical purposes. For example, calorie restriction without malnutrition while being highly effective at extending mouse lifespan is unlikely to provide proportional life extension in humans and might be difficult to follow [144]. Indeed, it is currently unclear whether calorie restriction in the true sense (i.e., not rescue from overeating/obesity) has any of the effects seen in rodents when it comes to humans.

Taking these considerations into account, one strategy could be to combine therapies that provided the highest lifespan extension as individual treatments. In descending order these would be (VEGF gene therapy up to +49% median), methionine restriction (up to +42% mean), TERT gene therapy (up to +41% median), follistatin gene therapy (+32% mean) and bone marrow transplantation (up to +28% mean). Note that the provided value of increased lifespan expectancy for bone marrow transplantation might be inflated given the high mortality in one of the studies’ control groups. A more conservative result of +12% median lifespan increase was reported in a more recent study [64].

Another strategy is to select the smallest number of treatments that cover the largest number of aging hallmarks (while still preferring treatments with highest lifespan extension values). For example, dasatinib + quercetin (CS + inf), rapamycin (DM + LP + DNS), VEGF gene therapy (AIC), bone marrow transplantation (SCE) and telomerase gene therapy (TA), perhaps with the addition of acarbose. Unfortunately, as mentioned earlier, we were unable to identify interventions that have confirmed positive effects on lifespan and can be applied to adult animals for several hallmarks of aging.

A third strategy is to start with the best existing combinations and expand them with interventions that provide the highest lifespan extension as an individual treatment but were not used before, preferably targeting a hallmark of aging that the initial combination did not. In this case we would recommend starting with rapamycin + acarbose (up to +37% lifespan extension) followed by the individual combinations from the first strategy.

A fourth strategy may be based on systematic screening for the synergistically best pairs of combinations that target different hallmarks of aging. This approach might also elucidate conflicting impacts of interventions that can offset or even worsen each other’s effects. The absence of an additive and synergistic effect may indicate, in particular, that the downstream effects of two therapies overlap.

Finally, a fifth strategy might consider investigating combinations that consist of widely-used molecular components with well-documented safety profiles in humans. Such combinations might include acarbose, glycine, taurine, and spermidine.

It’s worth noting that some combinations might be considered more practically feasible in real-life clinical settings. For example, small molecule drugs and supplements are easier to use than gene therapies, followed by bone-marrow transplantation. However, the widespread use of gene therapies by the general public is possible, given the availability of COVID vaccines which are similar in design. Multiple gene therapies have successfully finished human clinical trials and are available for a wide range of diseases from hemophilia A and B, to RPE65-mutation-associated retinal dystrophy, dystrophic epidermolysis bullosa and certain types of cancer [145]. Given the large amount of genes potentially involved in aging such as those reviewed in comprehensive Open genes database [146], we believe gene therapies are desirable and viable components of future anti-aging combination therapies.

Further directions

We observe that a number of genetic mutations increase the lifespan of mammals, but the effects of these mutations were not tested as possible longevity enhancing gene therapies. This includes gene therapies to overexpress Sirt6, Sirt1, p53 (+ p16 + Arf), FoxM1, catalase OE in peroxisomes + superoxide dismutase and the reduction of expression of GH, GHR, GHRHR, Prop1, mclk, macrophage inhibitory factor, IGF1R and Becn1F121A/F121A mutation in beclin 1. Individual testing of these approaches could provide novel single interventions that could be combined with future effective combination therapies. The combination of FGF21, αKlotho and sTGFβR2 deserves deeper investigation in terms of lifespan prolongation as a promising combination gene therapy that uses a number of genes that were all previously described as healthspan- or lifespan-promoting [51, 71, 147, 148].

There are some genetic and pharmacological interventions that are promising but, to our surprise, have not been tested in mice. One of them is the upregulation of FOXO3a, which is linked to human longevity and healthspan [149–152]. Some existing FOXO3a gene variants are associated with extreme longevity in human populations [149]. It is also known to be a positive regulator of healthspan, that affects the aging of the cardiovascular system [153]. The lifespan-expanding function of FoxO proteins is highly evolutionary conserved: the single FoxO homolog in Hydra contributes to the anti-aging mechanisms of this freshwater polyp [154, 155]. The prolongevity effects of homologous genes have been vastly studied in C. elegans, where they are associated with the DNS HA [156]. FOXO3 mediates the life-extending effect of CR in mice [114], but we haven’t found any studies where it was specifically overexpressed in mammals and lifespan changes were assessed.

Limitations

One important limitation of our analysis is that the effects of treatments on lifespan or other aging-related metrics in mice are at least to some extent dependent upon the conditions in which the animals are maintained and the genetic background of the strains used. Long-term breeding in laboratories has led to some strains being mildly progeroid, thus some of the life-extending effects could result from the rescue of the underlying conditions [157, 158]. This may further reduce the translatability of pro-longevity treatments in mice to other animals including humans. Additional studies in other species, such as dogs, could help elucidate the pro-longevity treatments that are more generalizable.

Another limitation comes from the hallmarks of aging. While currently we do not have a better framework for describing the aging process, the hallmarks of aging have relatively limited explanatory value regarding the general etiologies of aging and do not discern the primary and secondary causes of aging [20]. As we understand more about the aging process, we will be able to provide a better framework for selecting the most efficacious combination treatments.

There is also the question whether aging can truly be intervened. We believe it is possible given that the lifespans of many model organisms ranging from roundworms to mice have been greatly improved. The potential for human life extension is also supported by the increase of human lifespan during the course of evolution, when compared to our ancient ancestors. Finally, the existence of negligible senescence in some mammals, such as the naked mole rat suggests that aging is not an insurmountable challenge for life [2].

Author Contributions

All authors discussed and conceptualized the ideas, participated in writing and literature search, and approved the final manuscript. Additionally, AO and ASB contributed to the tables, AE and MB contributed to the figure, and TG and AYP drafted the final version of the text.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Funding

This work was supported by the Open Longevity CA nonprofit organization, along with Shurick Agapitov, Alexey Kovalev, and Artem Rudi.

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