Removal of Senescent Cells as treatment of age related Disease

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1. Role of Senescent cells in Aging

A. What is Aging?

"The  greatest trick the devil ever pulled was convincing the world he didn't exist."


Aging is an extremely complex disease and the entire group of age-related diseases can be seen as complications of aging.  Senescent cells and elevated mTOR play a major role in aging and age-related disease.


My first encounter with senescent cells was some forty plus years ago with the sudden onset of  severe back pain and muscle spasm. The  condition resolved in a few hours. The  sudden back pain was caused by a herniated lumbar disc. I recently came to understand the deteriorated of the disc was caused by the action of  senescent cells and their secretion of  chemicals (SASP) which caused  the deterioration of the glycosaminoglycans which comprise the extracellular matrix of the lumbar intervertebral disc. [Most everybody has experienced a similar event; the first dramatic sign of aging.]


It is widely proclaimed that "no one dies of old age or aging". In reality almost everybody dying of natural causes over the age of 60 is dying from aging. This is true whether dying of  heart disease, cancer or COVID-19. (If you were 20 years old, you would not die of these disease.) 


One can describing aging in  mathematical terms. The risk of death increases as an exponential curve with age.

 

If  people were immortal, in the sense of not aging, the risk of death would be a straight line for all adults. 


The impact of aging can be seen in actuarial tables which show the probability of dying in one year:


Exact age        Probability of dying in one year

20                                      .001

30                                     .002

40                                     .0024

50                                     .005

60                                     .01

70                                     .02

80                                     .06

90                                     .16

100                                   .35


The above table shows the risk of death in one year is 350 fold greater for a 100 year old compared to a 20 year old. 


Whether one looks at cells, tissues, organs, specific diseases or risk of death; the impact of aging is all too clear. 


Aging kills by age-related disease. Aging and age related disease are merely different manifestations of the same disease process. If not for aging; there would not be age-related disease.


At the center of Aging is elevated mTOR and senescent cells. 


The purpose of this website is to provide an in depth examination of the part played by senescent cells in Aging and age-related disease. The website also undertakes a detailed examination of senolytics, which are drugs which remove senescent cells.


There is now  a sufficient body of evidence  to justify the introduction of SENOLYTICS into clinical  medicine. 



B. The Treatment of Aging

Physicians should treat aging for the following reasons:

a. Aging meets the definition of disease. A disease is defined as: 

"A disorder of structure or function in a human, animal, or plant, especially one that produces specific signs or symptoms or that affects a specific location and is not simply a direct result of physical injury." 


b. The treatment of aging generally requires the use of potent drugs such as rapamycin and dasatinib; only available by prescription.


c. Aging is extremely complex; certainly as complex as any other disease.


d. Aging and age related disease cause great pain and suffering.


e. Regardless of whether aging is considered a disease or not; aging is easy to treat.


Aging can be divided into two very different mechanisms: 

 (1) natural aging 

 (2) active aging. 


Driving with one foot on the gas and one foot one the brake is analogous to natural aging and active aging. 


In natural aging, the body does its best to repair the damage which randomly accumulates with time. 


In active aging, the organism's own actions causes damage, decline and death. Active aging is a process that goes back countless millions of years and is embedded in our DNA by evolution.


Passive aging is the classic concept of aging. In simple terms it is failure of good housekeeping. As a result of wear and tear there is random damage. As a consequence of failure of good housekeeping to maintain complete repair, there is slow accumulation of damage. This type of aging is analogous to that seen in inanimate objects like an aging car. The damaged parts include mitochondria, DNA, nuclear membranes etc. This type of damage does not present as classic age-related disease.   


Active aging is aging driven by mTOR and senescent cells.  Active aging is a new concept and not part of established aging dogma.  


As discussed below, natural aging and active aging can interact. For example, damage to DNA (natural aging) can result in formation of senescent cells (active aging).


Active Aging:

In 2006, Mikhail Blagosklonny described how the mTOR pathway caused hyperfunction. In 2009 it was shown that rapamycin increased lifespan in mice by lowering the activity of the mTOR pathway. Since 2009, a very large body of basic science studies have shown that increased activity of mTOR1 is at the center of almost all age-related disease. 


Senescent cells were first described by Hayflick in 1961; however, it was not for another 50 years before their role in aging came into focus. The first clue to the role played by senescent cells is 2011 (Baker) discussed in Section 4. The first use of Dasatinib as a senoytic to kill senescent cells is 2015 (Zhu) also discussed in Section 4. The demonstration that senescent cells fulfills Koch's postulates showing senescent cells cause the physical dysfunction called Aging is discussed in 2018 paper. (Xu, Section 4.)


Because the  mTOR pathway and the senescent cell pathway are actively driving aging and age-related diseases through metabolic pathways; they present specific targets for  anti-aging treatment.


In mouse studies; lowering mTOR or removal of senescent cells increases lifespan and ameliorates age-related disease.


In the year 2020, these are the two major pathways to treat Aging. The major pharmacological methods to address these pathways are:


1. Rapamycin:  Lowering the mTOR1 pathway.

2. Senolytics: Removal of senescent cells. The leading senolytics are Dasatinib, Quercetin, and Fisetin.


The use of rapamycin to lower the mTOR pathway is discussed in detail in my website:   

                               www.rapamycintherapy.com


The focus of this website is the role of senescent cells in aging and age-related disease and the use of senolytics. 


As noted above, both the mTOR pathway and senescent cells can be called active aging. If a drug treats active aging it should meet the following two criteria:


1. A drug that prolongs life span in model organism preferably in mammals.

2. A drug that prevents several age-related diseases in mammals.

"From rapalogs to anti-aging formula," Blagosklonny, 2017.


Warning: The anti-aging field is overrun by Quack Medicine. Any claimed anti-aging remedy must meet two above criteria by Blagosklonny.


The list of diseases ameliorated by an anti-aging drug should include: atherosclerotic heart disease, Alzheimer's disease and cancer as these are the three leading age-related diseases.

 

In a recent paper, "Paradoxes of Senolytics", 2018, Blagosklonny writes:


"Senolytics are drugs that extend lifespan and delay some age-related diseases by killing senescent cells...Targeting senescent cells has been shown in animal models to prevent such age-related pathologies as emphysema, lung fibrosis, atherosclerosis, osteoporosis, osteoarthritis, renal disease, intervertebral disc pathology, hepatic steatosis." (since that paper, Alzheimer's disease can be added to list)


In the pathology of aging it seems like all roads lead to senescent cells. They appear to be the central hub in great many upstream and downstream pathways. The upstream pathways including elevated mTOR signaling, DNA damage, mitochondrial damage, oxidation stress, and short telomeres, which all lead to increase production of senescent cells. Downstream to senescent cells is the long list of age-related disease.


In addition, while elevated mTOR pathway is a major driver of aging and age-related disease, very many of the harmful actions of mTOR relate to senescent cells. mTOR accelerates the production of senescent cells and mTOR increases the production of the harmful chemicals call SASP that senescent cells produce. 


Sections 2-5 discuss the science papers showing that Aging and age-related disease is due in large part to the accumulation and harmful action of senescent cells.


At present, almost everybody dies from active aging. The great question is if we could decrease active aging, what would the natural aging landscape look like?

C. Pathology of Senescent Cells

In the pathology of senescent cells, three main characteristics can be identified:


1. Senescent cells are blocked from cell division and can't become two new cells. Cellular senescence is a state of irreversible cell-cycle arrest. This has a major impact on tissues which require stem cells to replace lost cells. In a 2018 paper (Lewis-McDougall) discussed in Heart portion of section V below, show how aged senescent cells contribute to age-associated cardiomyopathy. In 70 y/o subjects, over half of cardiac stem cells are senescent and can't form healthy new heart cells. This contributes to cardiac failure. 


2. Senescent cells cause a very damaging bystander effect in neighboring healthy cells.  In a 2012 paper, "A senescent cell bystander effect: senescence-induced senescence," (Nelson) argued "continuous exposure to senescent cells induce senescence in intact bystander fibroblasts. Thus senescent cells can induce a bystander effect, spreading senescence towards their neighbors." This effect is similar to the well known phenomena described in the old expression: "one rotten apple spoils the barrel."


In the 2018 Xu study discussed in Section 4 and 5, they showed that injection of a small number of senescent cells in young mice had a profound effect in both induction of physical dysfunction characteristic of frailty and five-fold increased risk of death. They also confirmed spread of cellular senescence into host tissues. They concluded that this spread of senescence explained why transplantation of a very small number of senescent cells, which would only survive up to 40 days, could have such a profound effect on physical dysfunction and decrease  in survival.


3. SASP:  This is abbreviation for senescence-associated secretory phenotype. This term refers to the witches brew of highly active substances produced by senescent cells. This consists of a fearsome mixture of pro-inflammatory cytokines, chemokines and proteases. 


The current study of age-related diseases is to a large degree, the study of how these various SASP phenotypes cause specific diseases. 


In the study of systemic sclerosis discussed in section 2; there were 28 SASP pathways and whether a patient improved was related to changes in various SASP levels.


In the Section V in section on Heart,  a 2016 paper by Childs showed how senescent cells  drive all features of atherosclerosis by secretion of various SASP factors. One example was destabilization of fibrous plaque by metaloprotease (MMP) production which dissolves collagen and caused rupture of plaque  with resulting bleeding and clot formation and arterial occlusion, which then presents as "heart attack."


To understand the extraordinary harmful effect of senescent cells requires a study of each age-related disease and the specific action of senescent cells; how senescent cells mold the pathology of the  disease.

D. Aging and Benjamin Gompertz (1825)

In 1825, Benjamin Gompertz presented a remarkable paper to the Royal Society of London.


Gompertz stated aging could be described as an exponential curve. When external causes of death were excluded, death from natural causes increases exponentially with age. The Gompertz law of mortality describes the age dynamics of human mortality rather accurately in the age window from about 30 to 80 years of age. At more advanced ages some some studies have found death rates increase more slowly. The curve was calculated to have a doubling of mortality every 8 years.


Explaining aging starts with the mechanism which best explains the Gompertz exponential curve. (1)


In 2006 study, "Cellular Senescence in Aging Primates"  (2) it was found that number of senescent skin fibroblast increase exponentially with age. 

5 years    1%

15 years   5%

25 years  15%

30 years  30-35%.


This same exponential increase in numbers of senescent cells is also found in mice. 


In the recent paper, "Senescent cell turnover slows with age providing an explanation for the Gompertz law," the authors constructed mathematical models to duplicate the senescent cell numbers in mice at various ages. The best fit they termed, "saturation of removal." The explanation for best fit is "senescent cells reduce their own removal rate." In this model, senescent cells increase with age and as they increase, they decrease their own removal rate, resulting in a faster accumulation.


This same mechanism has a good fit with humans; but humans have a 60-fold slower increase in senescent cell production compared to mice. The slower rate of senescent cell production rate in humans was attributed to improved DNA maintenance in humans compared to mice. In a study supporting this idea, the ability of various species to do DNA repair correlates with life-span of a species.


The following was in declining ability:

Excellent: Man, elephant, cow 

Poor: rat, mouse shrew.  [Hart 1974] (3) 


In mice senescent cell turnover (half-life) is fast  (5 days)  in young mice (3 months old); but slows to 25 days in old mice (22 months). 


The model suggest a type of vicious circle in which more senescent cells results in slower removal and faster accumulation.


 At a certain number (~30%) the animal appears to reaches a tipping point resulting in death. 


The deceleration of mortality rates at very old age is due to those with high levels of senescent cells having already died. 


The paper suggests that senescent cells cause death when their numbers exceed a threshold. Conclusion: "Our results suggest that treatments that remove senescent cells can have a double benefit: an immediate  benefit from a reduced  senescent cell load, and a longer-term benefit from increased senescent cell removal."


Note: In this model the abundance and health of immune cells is critical and at certain point, the demand exceeds the  ability of immune cells which can be the tipping point. 


"Similarly, interventions that increase removal capacity, for example by augmenting the immune surveillance of senescent cells are predicted to be an effective approach to reduce senescent cell levels. ."

[Note: rapamycin improves function of immune system in older people].


1.  Senescent cell turnover slows with age providing an explanation for the Gompertz law.Karin, 2019.


2. Cellular senescence in aging primates. Herbig, 2006


3. Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species. Hart, 1974. 



E. Clinical Considerations Regarding Senolytic Treatment

The 2019 Hickson paper, discussed at length to section II, concludes:


"In the current clinical trial we show that D + Q is effective in decreasing senescent cells (in humans)... Although we are optimistic about the prospects for introducing senolytics and other agents that modulate fundamental aging processes into clinical practice in the future, particularly in the near future for serious diseases for which there are currently no effective interventions, we must conclude with a note of caution... We caution against the use of senolytic agents outside the context of clinical trials until more is know about their effects and side effects."


If we waited for clinical trials to provide definitive evidence, the field of anti-aging would not exist. If we wait for clinical trials to be concluded it will deprive millions of people of the benefit of senolytics in their own lifetime. 


The purpose of this website is to present sufficient data so that patients can make their own informed decision regarding whether to use senolytics now or wait until more is known.


The main three senolytics I use are Dasatinib, Fisetin and Quercetin. Dasatinib is the generic name for Sprycel, made by Bristol-Myers-Squibb and approved since 1996 for the treatment of leukemia. Fisetin and Quercetin are natural flavinoids present in fruits and vegetables and sold as supplement and known to be very safe. Dasatinib has been approved in US for over 20 years and there is sufficient data about side effects. 


Regarding Dasatinib, there  are now three human studies and more than twenty animal studies regarding its role as a senolytic. It was apparent from those studies that the human results are basically comparable to animal results.


In five long-term mouse studies there was no apparent harmful effects from long-term removal of senescents cells. While more data may be needed to prove efficacy of senolytics and dasatinib in amelioration of age-related diseases and slowing aging in humans; there is not a scintilla of evidence suggesting harm from removal of senescent cells. 


A detailed protocol is presented in Section 6 of this website regarding clinical use of Dasatinib and other senolytics.



F. Programmed Aging and Anti-Aging Medicine,

Programmed aging is the foundation of modern anti-aging medicine. The leading expert on programmed aging is Theodore Goldsmith.  "Evolution of Aging Theories: Why Modern Programmed Aging concepts are Transforming Medical Research", Goldsmith, 2016. 


"Programmed (or adaptive) aging refers to the idea that humans and most complex organisms possess biological mechanisms that PURPOSELY limit their internally determined lifespans beyond a certain species-specific age and that these mechanisms are adaptations  in that they evolved because aging, per se, creates an evolutionary advantage. According to this concept, these senescent programs are ultimately responsible for most occurrences of highly age-related diseases and conditions such as cancer, heart disease, and Alzheimer's disease."


Currently anti-aging medicine targets two recently identified biologic mechanism that promote aging and age-related disease. These two processes are elevated mTORC1 and senescent cells. 


Biogerontology is the study of the biology of aging and at present most of  the "leading experts" in this field do not believe in programmed aging and do not believe in anti-aging medicine. Since anti-aging medicine is intimately linked to modifying programmed aging, it is natural that gerontologists that don't believe in programmed aging also don't believe in anti-aging medicine. 


[For this discussion, programmed aging and Blagosklonny's "quasi-programmed aging" is considered as essentially the same thing or at least having a distinction without a  difference.]


A central pillar of biogerontology is the evolutionary theory of aging. Darwin's survival-of-the-fittest evolutionary mechanics concept is that if an inheritable trait produces an INDIVIDUAL benefit resulting in an increased probability of producing descendants, this is individual fitness and will increase. Aging and death produce no individual benefit. Therefore, under classic Darwin theory, programmed aging is deemed  to be theoretically ruled out and dismissed as scientifically impossible. However, the problem is not with Darwin's theory of evolution; but rather with the faulty application.


Darvin's theory applies to the individual.  Aging and senescence produces a population benefit but at  a cost to the individual. The mechanism by which traits that benefit the group; but harm the individual can evolve may be outside classic Darwinian theory. However, it is certainly not outside computer programs of population dynamics. 


Even the great Mikhail Blagosklonny in his immortal paper, Aging and Immortality" 2006 argued:

"Programmed aging is questionable on theoretical grounds":

"There is a strong argument that a program for senescene simply cannot exist. As discussed previously from evolutionary point of view, there can be no special program that is intended to impair animal functions, to cause decline and deaths." 


David Gems is a leading gerontologist and proponent of how programmed aging is impossible. In paper by Gems he writes; "Does senescence promote fitness in C. elegans by causing death?"

"An important conclusion from evolutionary theory is that...senescence serves no purpose, i.e., it does not contribute to biological fitness (the capacity of an organism to spread its genes in a population). This means that there should not be genes whose naturally selected function is to cause aging".


However, the big question is always what happens when the rubber hits the road.


A 2020 paper by David Gems, "Shorter life and reduced fecundity can increase colony fitness in virtual C.elegan" answers this question. In this paper Gems goes outside the bubble of "classic evolutionary theory" and does an experiment. 


Abstract:

"These results imply that programmed, adaptive death could promote colony fitness in C. elegans through a consumer sacrifice mechanism (individual death). Thus, C. elegans lifespan may be limited not by aging in the usual sense but rather by apoptosis-like programmed death."


This paper is significant in that David Gems is a leading proponent of classic evolutionary theory; but when he does an actual experiment the findings show programmed aging promotes colony fitness. 


Genes that promote colony fitness for survival of the colony can be conserved over millions of years and become part of the standard genetic plan we all have.


Age-related disease can be seen as an atavistic disease. 


Twenty years ago, programmed aging was just an academic discussion. Now it is a topic of extreme importance. Between elevated mTOR in older animals and the discovery of the role of senescent cells; we now have two programmed aging mechanisms. Furthermore, we have a method to target both mechanisms. 


When the organism has a complex mechanism to promote aging; the organism has a target for anti-aging scientists. Furthermore, as everybody knows who has ever lived in an apartment and saw two cockroaches; there is always more than two.


At this point in time, we can now treat two fundamental causes of programmed aging. 

-----------------------------------

If senescent cells are not programmed aging they will do until the real programmed aging gets here. 


In Aging and Immortality", Blagosklonny 2006 writes:


"Why are we mortal?  No law of physics precludes immortality. Why then are we mortal?  As famously noted, 'it is truly amazing that a complex organism, formed through an extraordinary process of morphogenesis, should be unable to perform the much simpler task of merely maintaining what already exists'.'"


I would suggest that immortal organism did evolve; but they became extinct. They could not keep up with the never ending arms arms race. Immortality was a dead end for almost all the species that went down that pathway. Aging and senescence to maintain the ideal life span is the winning pathway discovered by evolution. 



2. Three Human Clinical Studies: Dasatinib + Quercetin

Hickson: 2019 "Senolytics decrease senescent cells in humans: "

"Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease" Hickson, Kirkland, 2019, EbioMedicine, Mayo Clinic, Rochester, Minn.


News Release 18-Sep-2019, Mayo clinic: 

"In a small safety and feasibility clinical trial, Mayo clinic researchers have demonstrated for the first time that senescent cells can be removed from the body using drugs termed 'senolytics.' The result was verified not only in analysis of blood but also in changes in skin and fat tissue senescent cell abundance."


This trial--involving participants who had diabetes-related kidney disease--is the second clinical  study of senolytics to be published by Mayo, but is the first to show that senolytic drugs, discovered by Mayo researchers, can remove senescent cells from humans as they did in numerous studies in animals."


"For three days the nine participants received a combination dose of dasatinib and quercetin. Though the drugs cleared the body in a couple of days, effects on reducing senescent cell were evident for at least 11 days. The researchers say this shows the senolytic drug combination significantly decreases senescent cell burden in humans. Senescent cells are characteristic in end-stage kidney failure as well as diabetes-related kidney disease. By removing the cells from mice, researchers had previously found that senolytics alleviate insulin resistance, cell dysfunction, and other processes that cause disease progression and complications."


"Senescent cells are malfunctioning cells that accumulate with aging and in organs affected by chronic diseases. Senescent cells can remain in the body and contribute to multiple diseases as well as features of aging, ranging from heart disease to frailty, dementias, osteoporosis, diabetes, and kidney, liver, and lung diseases."


"By targeting senescent cells with senolytics, we can delay, prevent, or treat multiple disease and increase health."


NIA deputy director: "The demonstration that senescent cell numbers can be reduced in human tissues in humans is an important advance based on the compelling evidence from studies in laboratory mice."

Hickson: 2019; First study to show Dasatinib would decrease senescent cells in humans.

Method: Dasatinib in dose of 100 mg plus Quercetin 1000 mg was given orally to individuals with diabetic kidney disease for 3 days. 11 days later the senescent cell burden was measured. The 3 day treatment was well tolerated.


Results:

1. Senescent cell in subcutaneous adipose tissue was reduced: 35%, 17%, 62% depending on marker used for senescent cells.

2. macrophages, which are anchored to senescent adipose cells were released and reduced 28%.

3. "Crown-like structures" (dead adipose cells) characteristic of adipose tissue in diabetes and obesity were reduced 86%.

4. Healthy adipocyte progenitors (stem cells) were increased 8%.

5. Senescent epidermal cells were reduced 20% as measured by length and 31% by marker.


6. Circulating SASP factors: IL-1a, -2, -6, -9, MMP -2, -9, -12  (Matrix Metalloproteinases) were significantly lower. 

Hickson: 2019; Discussion and Significance

D+Q which are senolytic in mice and tissue culture produced similar results in humans.


D+Q have elimination half lives of four and eleven hours. This study showed that a three-day course of treatment, "hit-and-run," reduced senescent cell burden for eleven days following the last dose. Senescent cells take weeks to over a month to form and acquire SASP, meaning that senolytic agents like dasatinib can be administered in a "hit and run" fashion. Most potential side effects can be avoided through intermittent administration for 3 days. In chronic leukemia the dose of 100 mg a day had been administered continuously for years.


Most SASP factors tested were significantly reduced. In another trial in humans there was a decrease in 53 out of 66 SASP factors with Dasatinib.


Discussion:  "[We suggest that] by targeting a fundamental aging process such as cellular senescence, multiple age-related disorders can be delayed, prevented or alleviated as a group, instead of one-at-a time." 


They also note that in mouse studies removal of senescent cells: improves cardiac function and vascular reactivity, alleviates frailty, increases intervertebral disc proteoglycans, alleviated age-related decline in cardiac regenerative capacity, muscle weakness, decreased osteoporosis, alleviated reduced running endurance on a treadmill. Additionally, in old mice, increased remaining lifespan by 36%. Also noted decrease in Alzheimer's-like dementia in mice. Also decreased fatty liver, and pulmonary fibrosis. 


AG comment: The results in mice show excellent anti-aging protection; so it's of great importance to show that senolytics works same in humans. The results of this human study were the same as would be expected in mice. 

Justice: 2019, First Human Study, Dasatinib in Idiopathic Pulmonary Fibrosis

"Senolytics in idiopathic pulmonary fibrosis: Results from a first-in-human, open-label, pilot study":, Justice, Kirkland, 2019, Mayo Clinic, Rochester, Minn. 


In this first-in-human pilot study published February 2019, Dasatinib was shown to alleviate physical dysfunction in Idiopathic Pulmonary Fibrosis (IPF) and increase 6-minute walking speed. 


The dosing was a 3 week course of intermittent dosing consisting of 100 mg of Dasatinib for 3 days plus Quercetin 1250 mg a day for 3 days; followed by a 4 day gap. This 3 day treatment was repeated for 3 consecutive weeks for total of 9 doses of Dasatinib 100 mg plus Quercetin. 


Results:

Physical function: 

6 minute walking distance

4 meter gait speed

Timed chair-stands

SPPB (mobility limitations)

Frailty index

All significantly improved.


Respiratory function:

Improved but not enough for significant number.


Overall: 6 patients had mild to moderate improvement. 


SASP factors:

Selected SASP factors including IL-6, MMP7. 

There were correlations with decrease in various SASP factor and improved physical function.


The improvement in function in human study was consistent with dasatinib and quercetin results in bleomycin induce pulmonary fibrosis in mice.


Conclusion: "In this first-in-human trial of senolytics, our data indicate that short-term, intermittent administration of D+Q may alleviate the physical dysfunction that accompanies IPF, as is the case with D+Q treatment of pulmonary fibrosis induced by bleomycin in mice."


AG: The critical significance is demonstrating that results in mice can be duplicated in humans

Justice: 2019, Dasatinib and Pulmonary Fibrosis study; Side Effects:

Patients characteristics:  Age, yrs (mean, range)  70.8  (55-84)

14 patients, (2 women). 


Reported adverse events:

Respiratory

Cough 6,  Moderate

Shortness breath 3, Mild

Running nose, sneezing 3,  mild

Respiratory infection/Pulmonary edema ? 1 severe


General health & well feeling:

Feeling generally unwell: 4

Tired/weak  4

Nonspecific dizziness, light-headedness 3, mild

Anxiety 2 


In general, the majority of the symptoms were mild to moderate, reversible, and without clinical significant sequelae. 


The most frequent events were respiratory symptoms reports: cough, shortness of breath, running nose or gastrointestinal discomfort or heartburn. 2 cases of headache. 

On serious event related to IPF was bacterial infection or pulmonary edema. 


No deaths.


"Reported adverse events were generally acceptable". Mild-to moderate severity diarrhea and nausea were most common.


The retention rate was 100% with no D+Q discontinuation. 


[My own impression was first 3 day course had runny nose, heartburn, diarrhea, feeling tired and unwell  especially 2nd day, all mild symptoms.


I repeated treatments once a month and by the third course of 3 day treatment, I had no symptoms with treatment.]

Martyanov: 2017, Human trial: Dasatinib; Systemic sclerosis-associated interstitial lung disease

"Novel lung imaging biomarkers and skin gene expression subsetting in dasatinib treatment of systemic sclerosis-associated interstitial lung disease"; Martyanov, 2017.

Dartmouth Medical School, Hanover, NH; UCLA Medical School, Los Angeles; South Carolina Medical School, Charleston SC.


"Senescence signature in skin biopsies from systemic sclerosis patients treated with senolytic therapy: potential predictor of clinical response?", Arthritis & Rheumatology, Oct 2019; Concise Communication.


This was open-label Phase 2a trial involving 31 patients with Systemic sclerosis (SSc) plus evidence of pulmonary fibrosis. All patients received Dasatinib 100 mg orally, daily for median length 9 months.


Of 31 treated, 8 discontinued, (5 adverse events)

23 were analysed. 


12 had pre and post skin biopsies and these 12 were subject to close evaluation of SASP markers.


Daily Dasatinib in dose of 100 mg for mean of 9 months was well tolerated. Most common adverse events were diarrhea and nausea.


Systemic sclerosis (SSc) is characterized by autoimmunity, widespread microangiopathy and multi-organ fibrosis. Progressive fibrosis in the skin and lungs account for significant morbidity and mortality of SSc. In the skin, excessive matrix deposition causes stiffening and tightening. In the lungs, fibrosis caused respiratory insufficiency associated with restrictive interstitial lung disease


Results: 

Dasatinib was well-tolerated in 31 patients for a median of 9 months. By quantitative HRCT (high resolution computed tomography) 65 % showed no progression of lung fibrosis and 39% showed no progression interstitial lung fibrosis.


Of  12 subjects with pre and post skin biopsies 3 were improvers and 9 were non-improvers. Improvers in skin biopsy also showed stability of lung fibrosis and non-improvers showed increase in lung fibrosis. 


The 2019 "Concise Communication" described the SASP signature in the skin biopsies.


The results showed that the SASP gene signature was significantly reduced from baseline in dasatinib-treated improvers; in marked contrast, no change from baseline was seen in non-improvers.


Among the 66 SASP signature genes 83% showed a decrease in expression post treatment in improvers, compared to only 53% in non-improvers


Heat map showed 28 SASP genes which showed significant difference in improvers and non-improvers. 


Conclusion: These results show clinically beneficial senolytic effect, suggesting that dasatinib-mediated clearance of pathogenic senescent cells in lesional tissue and consequent reduction in the systemic senescence burden may have mitigated the fibrotic process and led to clinical improvement.


"This mechanistic scenario closely parallels recent experimental observations in an animal model of bleomycin-induced lung fibrosis, where pharmacologic clearance of senescent cells using dasatinib plus quercetin resulted in function attenuation of the lung fibrosis. (Schafer, 2017).


AG comment:

1. Not a Mayo clinic study.

2. Patients took Dasatinib median 9 months daily and well tolerated.

3. Only used Dasatinib, no Quercetin.

4. Human results similar to mouse-model results. 


3. Life Span Extension Mice: Rapamycin, Senolytics

Studies in Middle Age and Old Mice: Extension Median Life Span

Study                    AGE Months        Treatment                     Males             Females  


Miller                             9               Rapamycin, low dose         13%                21%

                                       9               Rapamycin, high dose        23%               26%      


Baker                           12                    Senolytic                     23%, 35%         30% , 17%


Harrison                     20               Rapamycin, low dose            9%               14%   


Yousefzadeh             20                 Senolytic, Fisetin                        12% M+F


Xu                               24-27            Senolytic D+Q                             6% M+F


      



Discussion of Life Span Extension: Rapamycin

Miller: "Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction", Miller, 2014.

Harrison: Rapamycin fed late in life extends lifespan in genetically heterogeneous mice", Harrison, 2009.


In the Miller study and Harrison study the same dose was used in low dose part of Miller and Harrison. The young male mice had median lifespan extension of 13% compared to 9% for middle age mice. The young female mice female mice had 21% life span extension compared to 14% for middle age female mice. 

At 3 times the low dose the young male mice increased to 23% and young female mice to 26%.


Maximum life span extension required starting at a younger age than 20 months and required the proper dose. The male mice required a higher dose than female mice to achieve best results.


Age, sex and dose were critical factors for best results. 


Middle age mice had lower potential for life span extension than younger mice

Discussion of Life Span Extension: Senolytics

Baker, Yousefzadeh and Xu are discussed in detail in following section. 


One conclusion that should NOT be drawn from these studies is that the proper time to start senolytics as Fisetin and Dasatinib + Quercetin is OLD AGE. The studies were started in old age due to the prejudice of the persons doing the study that the proper time to treat old age is in old age and not for any scientific reason. The stated reason for starting in old age was that for purposes of translation into clinical practice. In the event there were any side effects they would not want young persons to have side effects. [In my opinion, that is politics; not science.]


In the  Baker study, they did not worry about translation into clinical practice. The method they used to eliminate senescent cells was a special genetically modified mouse which create the ability to remove senescent cells with a drug which did not have potential for human use. However, the results should be fully applicable to ordinary wild-type mice.


The Baker study is of extraordinary importance.


The Baker study revealed senescent cells start accumulating in middle age mice from 12 months to 18 months. 


When removal of senescent cells was initiated at 12 months; the extension of median life span was up to 35% and 30% in different groups of male and female mice. The overall results in different groups was: 27%, 24% (combined male and female), 23%, 35% (male), 30%, 17% (female). These were small groups and there was also issue of impact stress of frequent injections on lifespan. 


In the Yousefzadeh, Fisetin was senolytic used. Fisetin was started at 20 months which was same age as Harrison. Median life span extension was not reported; but could be calculated from figure of data as 12% (male + female combined). This appears to be in the same range as Harrison.


In the Xu study, Dasatinib and Quercetin was started at very old age of 24-27 months. Median extension of life span was NOT stated; but from figure appears to be about 6%.  As regards results they proclaim "increased post-treatment survival by 36%." However, in terms usually used in life span extension studies, it was about a 6% median extension of life span.


In the comparison between Fisetin compared to Dasatinib +Quercetin; Fisetin  had greater extension of life span. In a large number of studies, Dasatinib has been shown to be a very effective senolytic. The difference in results is best explained by difference in age of mice at which treatment started.


Analysis Rapamycin and Senolytic Life span Extension

These five studies are highly suggestive that aging is a one way street.


The interventions with both rapamycin and senolytics were able to slow aging. However, they did not appear to reverse aging. These studies point to the presence of irreversible changes.


These studies suggest that if you are a mouse looking for maximum life span extension you should start treatment in the 12 month to 18 month age range.


A 12 month mouse is equivalent to 40 year old human. 

4. Mouse studies: Senolytics and Health

2011 First mouse study, Progeroid type mouse

"Clearance of p16-Ink4a-positive senescents cells delays aging-associated disorders", Baker, Kirkland, Mayo Clinic, Rochester, 2011.

In this first study they used a progeroid mouse model with a transgene, called INK-ATTAC which allowed them to selectively remove senescent cells with administration of a synthetic drug (AP20187) . The targeted senescent cells accumulated in certain tissues including: adipose tissue, skeletal muscle and eye. 


The study showed that age related pathologies were related to accumulation of senescent cells. Removal of senescent cells delayed loss of muscle called sarcopenia, loss of adipose tissue and delay onset of cataracts. 


There were no negative side to clearance of senescent cells. 


The conclusion was: cellular senescence is causually implicated in generating age-related phenotypes and that removal  of senescent cells can prevent or delay tissue dysfunction and extend healthspan."


2018 Another Progeroid  Mouse Study: 

"Spontaneouss DNA damage to the nuclear genone promotes senescence, redox imbalance and aging", Robinson, Niedernhofer, 2018.


[AG: fascinating look at UPSTREAM  changes leading to formation of senescent cells]


Study uses progeroid type mouse called  Ercc. The Ercc mouse has only 5-10% of the normal complement of an endonucleas complex enzyme which repairs spontaneous breaks in  nuclear DNA. This is called nucleotide excision repair.  As a result, these mice accumulate oxidative DNA damage more rapidly than wild-type mice. The results show the Ercc mice have accelerated accumulation of spontaneous DNA damage due to  failure of nucleotide excision repair.  The mice then have an accelerated accumulation of senescent cells. The mice have greatly accelerated aging and very short life span. 


In this study they had used a drug to greatly reduce oxidative stress in mitochondria. The result was reduction in senescent cells and delayed onset of the age-related decline. These features included: Dystonia, trembling, kyphosis, ataxia, hind-limb wasting, urinary incontinence. 


[AG: The last line of conclusion and first line of Abstract read as follows:]


"In conclusion, we demonstrate that spontaneous, endogenous, nuclear DNA damage leads to an accelerated accumulation of senescent cells in vivo..."Accumulation of senescent cells over time contributes to aging and age-related diseases."


AG: Upstream to senescent cells there is DNA nuclear damage and downstream to senescent cells is age-related disease.

2015 Mouse Study, First Dasatinib + Quercetin use

"The Achilles heel of senescent cells: from transcriptome to senolytic drugs", Zhu, Kirkland, Mayo Clinic, Rochester, Minn, 2015.


This was a very important two part study. First part is identification of senolytic drugs and second part demonstration of action.


Summary: Identification of "new class of drugs termed senolytics, which selectively kill senescent cells."


Senescent cells have increase (upregulation) of prosurvival pathways which allow the senescent cell to resist APOPTOSIS.


This study selected drugs which targeted the genes which are anti-apoptosis and allow the senescent cell to survive. They show that if turn off the pro survival pathways of senescent cells; then the senescent cells undergo apoptosis and die. 


The two candidate drugs selected in this study were Dasatinib and Quercetin. Dasatinib preferentially reduced viability and caused cell death of senescent human preadipocytes (fat tissue) but was less effective against senescent HUVECs (ENDOTHELIAL CELLS) [human umbilical vein endothelial cells).


In contrast to dasatinib, Quercetin, a naturally occurring flavinoid, reduced viability and caused cell death of HUVEC, but was less effective against preadipocytes.

 

The combination of D+Q selectively killed preadipocytes and endothelial cells. 


D+Q also reduced viability of  FIBROBLASTS (MEFs, mouse embryonic fibroblasts).


D+Q also reduced number of MESENCHYMAL STEM CELLS (senescent bone-marrow-derived mesenchymal stem cells).


The best dose and ratio was 5 mg kg and 50 mg kilo for D+Q and was used in all studies.


Results:

D +Q given to old mice: Improved cardiac ejection fraction, vascular relaxation.


The conclusion was that the data suggests removal of senescent cells could improve cardiovascular function and reduce cardiovascular disease in elderly.


Prolonged action: One leg of 4 month old mouse was radiated to produced senescent cells. 3 months later mouse had reduced function with exercise in radiated leg. Given single treatment D+Q. 7 months after single-dose D+Q irradiated leg had same muscle function non-radiated leg.


Progeroid mouse:  A progeroid mouse which had accelerated aging was treated with D+Q, once a week, from age 4-6 weeks to age 10-12 weeks. 


Treated mice had following reduction in age-related pathologies:

kyphosis, dystonia, tremors, loss of grip strength, coat condition, urinary incontinence, impaired gait, hind limb paralysis, poor body condition.


In particular reduced dystonia, delayed onset of ataxia, gait disorders. Also improved bone parameters lumbar spine.


Increased proteoglycans in nucleus pulposus of the intervertebral disc. (The deterioration of intervertebral disc is cause of near universal back problems in older people)


Sections of liver, kidney, femoral bone showed less pathology. 


Conclusion:"Taken together, these data demonstrate  that periodic treatment with senolytics [D+Q] is sufficient to reduce the burden of senescent markers, reduce frailty, and extend healthspan significantly."

2016 Baker, Middle Age Mice, Healthy Lifespan

"Naturally  occurring p16-Ink4a-positive cells shorten healthy lifespan", Baker, 2016, Mayo clinic, Rochester Minn.


This is an extremely important study.  The focus of this study is 12 month to 18 month old mice. This is middle age mice; roughly the equivalent from 36 year old to 54 year old humans.In old age, animals have large numbers of senescent cells which cause clinical disease and death. In middle age the numbers of senescent cells are small and generally not causing significant clinical disease. Nevertheless, thse small numbers may be very important in the treatment of aging in the pre-pre-disease stage as discussed by Blagosklonny.


They used genetically modified wild-type mice with a "suicide gene" which would kill senescent cells when given a drug called AP20187 (AP). The senescent cells expresssed the marker called p16-Ink4a, a marker also known as Cdkn22a. The cells  had a green fluorescent marker for senescent cells. This is excellent model to track accumulation of senescent cells. They studied accumulation of senescent cells in adipose tissue, kidney and heart in the age range of 12 to 18 months. The drug AP was given twice a week, starting at 12 months old to continuously elimination senescent cells. At 18 months of age, mice are examined with focus on adipose tissue, kidney and heart tissue to determine effect of clearance of senescent cells in middle age.


Adipose tissue: 

Very few senescent cells were present in 12 month old mice (36 years). 


At 18 months  (54 years) there was an 8-fold  increase in senescent cells in adipose tissue from 0.2% of cells to 1.6% of cells in AP treated to untreated mice. 


The senescent cells were adipocyte progenitor cells which are cells which have potential of forming new adipose cells. 


What is most important is this relatively small number of 1.6% of senescent cells was sufficient to change the function of the adipose cells. In the mouse, the presentation was age-related lipodystrophy. Clearance of senescent fat progenitor cells attentuated the age-related lipodystrophy. The important thing was the adipose tissue had abnormal function.


In humans, senescent adipose tissue is insulin resistant. In my opinion, senescent fat cells and insulin resistance is first step on the path to middle age obesity.


Kidney tissue: At 18 months renal tissue showed 1.2% senescent cells compared to 0.3% in AP treated cells. At 18 month  the senescent cells in untreated kidney showed development of glomerulosclerosis and renal dysfunction. Surprisingly, the senescent cells were not located in the glomeruli but in the proximal tubules. The senescent proximal tubules resulted in overactivation of the RASS  system (renin-angiotensin system) which resulted in glomerulosclerosis.


Cardiac: Heart tissue showed increase in senescent cells in in untreated 18 month old mice. 


At 18 month the untreated mice analysed by echocardiogram appeared normal. They showed no difference in heart rate, left ventricular mass, ejection fraction all unchanged from 12 month old mice. 


It was only on histologic exam that differences could be detected. "Cardiac aging at the histological level is characterized by a loss of ventricular cardiomyocytes due to decreasing ability to replace [lost] cardiomyocytes, inducing hypertrophy of the remaining cardiomyocytes." 


The cardiac wall had the same thickness, but they had less cells. The treated mice had smaller heart cells but more cells. 


The conclusion was senescent cells were the key driver of age-related cardiac aging. Cardiac aging was a loss of heart cells and hypertrophy of remaining cells.


Cardiac aging was characterized by decreasing stress tolerance. Decreasing stress tolerance was showed by less stress  required to produce fatal cardiac arrhythmia. Less tolerance to stress was show by increase in cardiac hypertrophy in response to chronic non-fatal stress. 


Conclusion: At 18 months senescent cells are causing impaired function of vital organs adipose tissue, kidney and heart. 


Also noted: elimination of senescent cells was not associated with any overt detrimental effects. 


Conclusion: "Accumulation of senescent cells during adulthood negatively influence lifespan and promote age-dependent changes in several organs and their therapeutic removal may be an attractive approach to extend healthy lifespan."



2018 Old Mouse, Dasatinib: Transplants, Lifespan, Health

"Senolytics Improve Physical Function and Increase Lifespan in Old Age", Xu, Kirkland, Mayo clinic, Rochester, Minn, 2018.

Dasatinib + Quercetin

First part of study is transplanting small numbers of senescent cells. This study is suggestive of Koch's postulates, formulated in 1884. Koch created four criteria designed to establish causal relationship between a microbe and disease. By extension this study seeks to show causation between senescent cells and age associated physical dysfunction, called Aging.


1. Transplanting small number of senescent cells is sufficient to induce physical dysfunction in young mice.

The transplant was an intraperitoneal transplant of senescent fat cells called senescent preadipocytes into 6 month old wild-type mice. 

They calculated that dose of transplant cells was 0.5 million cells. This would be only 1 cell in 7,000 to 15,000 throughout the body or 1 cell in 350 cells locally transplanted senescent cells. As early as 2 weeks after transplant mice developed old-age signs of frailty including decreased walking speed, hanging endurance, muscle strength. It was estimated the transplanted senescent cells would only survive 40 days. However, physical impairment persisted for up to 6 months. Two months after transplant the mice had senescent cells in visceral fat that was native senescent mouse cells not transplanted cells. The mice had an increase in SASP factors higher in muscle tissue.  

They also transplanted same number of senescent cells into 17 month old mice. The older mice had a much more severe physical impairment then the younger mice. Over the following year, the 17 month old mice that received senescent cell transplant had 5 fold higher risk of death. 


D+Q administration both prevents and alleviates the physical dysfunction induced by senescent cell transplant. D+Q at same time of transplant prevented physical dysfunction. D+Q 5 weeks after transplant  attenuated physical dysfunction from transplanted cells. 


Summary states: " Here we demonstrate that transplanting relatively small number of senescent cells into young mice is sufficient to cause persistent physical dysfunction, as well as spread cellular senescence to host tissue. Transplanting even fever senescent cells had the same effect in older recipients, accompanied by reduced survival, indicating the potency of senescent cells in shortening health and shortening life-span." The senolytic cocktail, dasatinib plus quercetin, which causes selective elimination of senescent cells, alleviated physical dysfunction and increased post-treatment survival in senescent cell transplanted mice.


[Second part study]

"Clearance of senescent cells alleviates physical dysfunction and increases late-life survival without extending morbidity in aged mice."


They treated 20 month old mice with D+Q for 4 months bi-weekly (once every two weeks).

The D+Q treated mice had improvement in maximal walking speed, hanging endurance, grip strength and treadmill endurance. All these were markers of increased muscle strength and less frailty.


The conclusion was: "Intermittent administration of oral administration of senolytics to ...naturally-aged mice alleviated physical dysfunction." 

2018 Old Mouse, Fisetin: Lifespan, Health

"Fisetin is a senotherapeutic that extends health and lifespan", Yousefzadeh, Kirkland, Mayo clinic, Rochester, Minn, 2018.

Fisetin

Results:

1. Fisetin in vitro reduced senescent cells of species: 

MEF (murine embryonic fibroblasts;

HUVECs (human umbilical vein endothelial cells) 

2. Progeroid mice, ages 6 to 14 weeks of age fed daily Fisetin 60 mg/kg daily reduced senescent cell burden.

3. Progeroid mice from 10 weeks of age to 20 weeks fed Fisetin 60 mg/kg and then tissues collected. Fisetin reduced SASP in all tissues, and CD3 T cells, in peripheral blood.

[AG note: CD3T cells demonstrates a robust increase as humans age and has been proposed as marker of human aging.]

Fisetin reduced oxidative stress in liver as measured by lipid peroxidation and increase in ration of reduced to oxidized glutathione {GSH: GSSG].


4. To confirm effect on progeroid mice, studies wild-type mice. Fed Fisetin 100 mg/kg for 5 days in 22-24 month old mice, sacrificed 3 days after last dose.


The 24 week old mice in fat tissue had significant increase in senescent cells identified as mesenchymal stem cells, T lymphocytes, natural killer cells, endothelial cells. Significant reduction of multiple cell types by fisetin.


In human adipose tissue reduced SASP factors: IL-6, IL-8, MCP-1.


5.  Lifespan and health:

Wild-type mice fed Fisetin, 60mg/kg starting 85 weeks equivalent 75 years in humans:

"This resulted in extension median as well as maximum lifespan." [AG discussed other section]


Tissues: Brain, kidney, liver, lung, forepaw showed reduced age-related pathology.


Fisetin reduced SASP and senescence markers, markers of oxidative stress [GSH:GSSG ratio].


[AG: This study established Fisetin as robust senolytic.]





5. Senescent cell Removal in Animal Models of Disease

1. Heart and Cardiovascular System

Zhu 2015: Senolytics improved cardiovascular function, D+Q improved cardiac ejection fraction. 


Baker (2016) Removal senescent cells delayed cardiac aging and cardiomyocyte hypertrophy with loss of heart cells, prevented development of stress intolerance. 


"Aged-senescent cells contribute to impaired heart regeneration". Lewis-McDougall, 2018.


D + Q study: human cell culture, wild-type mice


For the heart to continue to function heart stem cells called cardiac progenitor cells (CPCs) must continue to replace lost heart muscle cells. Failure to replace heart muscle cells results in increase in size of remaining cells (cardiac hypertrophy) and fibrosis on the path to death from heart failure. 


In study over half of CPCs in human subjects over 70 were senescent. The senescent cells can't regenerate into new heart cells. Even worse, senescent CPCs secrete SASP which render  otherwise healthy CPCs to senescence. Global elimination of senescent cells in wild-type mice using D+Q  in vivo activates resident CPCs and increased number of small new  cardiomyocytes.

 

Results:

1. CPCs exhibit a senescent phenotype with increased age.

2. Aged-senescent CPCs lose their regenerative capacity.

3. Aged-senescent CPCs secrete SASP  and elimination of senescent CPCs using senolytics D+Q abrogates SASP.

4. Wild-type  aged mice 24-32 months given D+Q in 4 cycles for 3 consecutive days, 12 days apart (once every 2 weeks)  showed increase in numbers of small proliferating cardiomyocytes in the aged heart. Also decrease in fibrosis.


"These finding show that clearance of senescent cells leads to stimulation of CPCs and cardiomyocytes with increased DNA-synthesizing activity and this strategy is specific to the aged heart."


"In conclusion...approaches that eliminate  senescent cells may be useful for treating age-related cardiac deterioration and rejuvenating the regenerative capacity of the aged heart." 


AG comment: the importance of this paper can not be overstated. It shows how senescent cells can produce an age-associated cardiomyopathy by reduction of  replacement of  dying heart cells. This same effect was shown in Section 4, Baker, 2016.

----------------------------------------------------------------------------------------

"Senescent intimal foam cells are deleterious at all stages of atherosclerosis", Childs, 2016.

 

This study used an atherosclerosis prone mouse model  and combined technique to remove senescent cells. The study showed senescent cells are  the key driers in all stages of atherosclerosis. 


"In advanced lesions, senescent cells promote plaque instability, including elastic fiber degradation and fibrous cap thinning, by heightening metalloprotease (MMP) production."


AG: In this extremely important study they  showed the pathogenesis of atherosclerosis from beginning atheroma to heart attack. They did not use D+Q or an available senolytic in clinical medicine. However, rapamycin has same target of reducing senescent cell production, SASP and reducing MMP.  This is very important study showing role of senescent cells in pathogenesis of atherosclerosis.


______________________________________________________________________

"Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice, " Roos, 2016.


D + Q mouse study.

 Aged mice treated once monthly for 3 months. Mice with advanced atherosclerotic lesion treated once weekly for 4 months. 

D+Q reduced senescent cells in medial layer aorta and reduced calcification.

Did not reduce senescent cells in intimal plaque, possible due to lack of penetration. 

2. Alzheimer's Disease, Neurologic disorders

Three recent papers present extraordinary new insight into Alzheimer's disease. 


The modern view of Alzheimer's disease is that it is a two-stage process. The first stage is  deterioration of the cerebral microcirculation.  This first hit is shown to be prevented by rapamycin in animal models. (www.alzheimer-prevention.com) 


The second stage is neuroinflammation which spreads from the hippocampus through cortex is a heretofore mysterious manner. The three main players in the inflammatory diseases process are neurons with intracellular inclusions called neurofibrillary tangles (insoluble tau)(NFT);  beta-amyloid extracellular plaques and glia which are astroglia, microglia, oligodendrocytes, and oligodrodrocytes precursors. 


Prior to the understanding of role of senescent cells, the neuroinflammation was very poorly understood. These three papers shed great light on neuroinflammation. 


Senescent cells appear to be the drivers of neuroinflammation. While these three papers discuss Alzheimer's disease, it is quite possible that senescent cells are the driving force  behind the neuroinflammation in all the neuropathologies with abnormal inclusion bodies. This includes Parkinson's disease, Fronto-temporal encephalopathy, and Huntington's disease. 


"Senolytic therapy alleviates AB-associated oligodendrocyte progenitor cells senescence and cognitive deficits in an Alzheimer's disease model", Zhang (NIH, Baltimore) 2019.


"Tau protein aggregation is associated with cellular senescence in the brain", Musi, 2018, Barshop Institute, San Antonio,Texas.


"Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline", Bussian, Baker, 2018, Mayo Clinic, Rochester, MN.


The Zhang paper describes senescent oligodrodrocyte precursors which when associated with extracellular beta-amyloid plaques become senescent and drive neuroinflammation.


The Musi paper describes how neurons with with insoluble pathogenic Tau called NFT become senescent neurons and drive disease.


The Bussian paper describes an early stage before cognitive decline with senescent astrocytes and microglia, forming near hippocampus and then driving diseases and  promoting formation of NFT in neurons.


The causation of the brain destruction in Alzheimer's appears to be due to these three groups of senescent cells. Removing these senescent cells ameliorates the disease.


Zhang: Dasatinib+Quercetin reduced senescent oligodendrocyte progenitor cells, reduced beta-amyloid plaque in hippocampus, reduced neuroinflammation and improved memory and learning. 


Bussian: Navitoclax (not yet approved drug) was used as senolytic. Early accumulation of senescent astrocytes and microglia in hippocampus was studied. These senescent cells promoted formation of insoluble tau aggregates (NFT). Removal of these senescent cells with navitoclax prevented NFT and preserved cognitive function. 


Musi: D+Q senolytic. The neurons with with NFT become senescent cells. D+Q treatment reduced NFT burden and  mediated neuroprotection.


In animal models, rapamycin acted on the first stage and could  prevent development of AD, but senolytics are acting on second stage and ameliorating established disease. 


Neurologic Disorders:

Zhu 2015: In progeroid mouse, D+Q reduced neurologic disorders Including: ataxia, dystonia, tremors, impaired gait, hind leg paralysis.

3. Lung, Kidney, Liver Disease

LUNG: Pulmonary Emphysema, Pulmonary fibrosis

"Cellular senescence as a mechanism and target in chronic lung diseases", Barnes, 2019.

"Cellular senescence is now considered an important driving mechanism for chronic lung diseases." [Excellent review]


Pulmonary emphysema:

"Elimination of p19-ARF expresssing cells protects against pulmonary emphysema in mice", Kikawa, 2018.


Navitoclax mouse study

The major cause of emphysema is smoking which causes production of senescent cells. Senescent cells then cause emphysema. The mechanism is production of SASP; which attracts macrophages which secrete MMP-12 also known as macrophage elastase, The elastase causes destruction of alveolar walls leading to permanent enlargement of airspace. 


This study used mouse model of emphysema  and used Navitoclax  to eliminate senescent cells. The elimination of senescent cells prevented lung tissue from elastase-induced lung dysfunction which depends on reduced pulmonary inflammation.The conclusion was 'the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema-associated pathologies. However, alveolar collapse is considered to be irreversible. Conclusion: "the presents results imply that senescent cells exacerbate pulmonary emphysema and have potential as a therapeutic/preventative target for emphysema. 


Idiopathic Pulmonary fibrosis: 

"Cellular senescence mediates fibrotic pulmonary disease", Schafer, 2017.


D+Q mouse study

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with median onset at age 66 and estimated 3-4 year survival. The disease shows markers of senescent cells and removal of senescent cells rejuvenates health and disease. Early intervention with a D+Q cocktail improves physical function and physical health. 


Results show:

1. Senescent biomarkers accumulate in IPF lungs. 

2. The secretome (SASP) of senescent fibroblasts is profibrotic.

3. Senescent fibroblasts are eliminated by D+Q.

4. Senescent cell clearance by D+Q mitigates fibrotic lung disease.


Discussion:  "Our murine results strongly support the hypothesis that senescent cell elimination potently influences health outcomes when animals are treated in early-stage pathogenesis, highlighting the potential utility of senolytics a an intervention strategy to be paired with early disease detection.".."Our findings causally implicate senescence and the SASP in fibrotic lung disease, thereby revealing senescent cell elimination and SASP blockade as novel therapeutic approaches for the treatment of IPF."

  

KIDNEY:

Baker (2016). Removal of senescent cells ameliorated renl epithelial senescence and development of glomerulosclerosis.


LIVER:

"Cellular senescence drives age-dependent hepatic steatosis", Ogrodnik, 2017.  D+Q study


Study used a rat model that develops non-alcoholic fatty liver.  A combination of D+Q removed senescent cells and reduced fat accumulation. Producing senescent cells increased fat. Mechanistically, the mitochondria lose ability to metabolize fatty acids efficiently. Clearance of senescent cells decreases fat accumulation. Severity of NAFLD correlates with burden of senescent cells.

4. Osteoporosis, Osteoarthritis, Intervertebral disc

OSTEOPOROSIS

"Targeting cellular senescence prevents age-related bone loss in mice." Farr, 2017.


Treatment of 20 month old wild-type mice with Dasatinib and Quercetin once monthly for 4 months prevents age-related bone loss. D+Q improved bone microarchitecture, increase cortical thickness and bone strength, reduced percent senescent osteocytes.


OSTEOARTHRITIS:

"Local clearance of senescent cell attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment," Jeon, 2017.


In mouse model of osteoarthritis, clearance of senescent cartilage cells attenuated development of arthritis, reduced pain and increased cartilage development.


"Transplanted senescent cell induce an osteoarthritis-like condition in Mice". Ming Xu, 2017.


Transplanting senescent cells into knee region caused leg pain, impaired mobility and x-ray and histological changes suggestive of osteoarthritis. Nonsenesent cells has less of these effects. 


Intervertebral Disc pathology:

Zhu (2015): Treatment progeroid mouse model with D+Q: Level of proteoglycans in nucleus pulposus of the intervertebral disc, a marker of disk degeneration, was significantly increased in D+Q treated mice. 

5 Adipose tissue, Insulin resistance, Overweight, Obesity, Metabolic Syndrome, Diabetes,

Baker, (2011) Maintenance of normal fat tissue.

Baker (2016) Removal senescent cells attenuates age related lipodystrophy.


Causation of Increase Abdominal adipose tissue after after 40:

Increase senescent cells in visceral adipose tissue --> dysfunction of adipose tissue, Insulin resistance --> increase insulin output from Beta cells Pancreas --> Increased mTOR  --> increasing amounts  adipose tissue --> overweight -->  Obesity --> acceleration of Aging, increased risk all age-related diseases, increased risk Diabetes. 


Treatment Targets: Reduce senescent cell burden adipose tissue, reduce mTOR


Obesity is disease of dysfunction of adipose tissue similar to the way heart disease, lung disease, kidney disease is the dysfunction of those organs. 


The marker of diseased adipose tissue is insulin resistance. The test for Insulin Resistance is elevated HOMA-IR score computed from fasting blood sugar and fasting insulin same blood sample.


"Targeting senescent cells alleviates obesity-induced metabolic dysfunction"; Palmer, Kirkland, 2019.


Dasatinib + Q study.


Results:

1. Senescent cell accumulate in visceral fat in obesity.

2. Senescent cell clearance improves insulin sensitivity.

3. D+Q reduced senescent cell burden, improved insulin sensitivity. 


D+Q: administered 5 consecutive days monthly or 3 consecutive days bi-weekly.


6. Stem Cells, Frailty, Eyes, Cancer

Stem Cells:

"Clearance of senescent cells by ABT263 rejuvenates aged hematopietic stem cells in mice", Chang 2016.


The drug used ABT263 is Navitoclox, a cancer drug not available. It is inhibitor of anti-apoptotic proteins BCL-2. In study elimination of senescent hematopoietic stem cells or senescent muscle stem cells was beneficial as rejuvenated the non-senescent aged stem cells. 


Muscle, Sarcopenia ,Frailty: 

Baker, 2011 decrease in loss of muscle, sarcopenia.


"JAK inhibition alleviates the cellular senescence associated secretory phenotype and frailty in old age." Ming Xu, 2015.


The drug used was Ruxolitinib, a very expensive cancer drug. Study showed treatment of aged mice for 10 weeks eliminated senescent cells from fat tissue and enhanced physical function characterized as frailty.


"Cellular senescence biomarker p16--INK4a Cell burden in Thigh adipose is associated with poor physical function in older women", Justice, 2017.


This study counted sensencent celll in thigh adipose tissue in overweight/obese older women. After adjusting for age and obesity, the abundance of senescent cells was directly related to frailty as measured by physical function, walking speed, grip strength. 

(ASG comment: It's not just mice).


Xu (2018)

"D + Q alleviated physical dysfunction, with higher maximal walking speed, hanging endurance, grip strength, treatmill endurance and daily activity in mice treated with D+Q compared to Vehicle."


Eye: Baker (2011) decrease in cataracts.  

Baker (2016). Removal of senescent cells delayed cataract formation.


Cancer:

Baker (2016) Removal senescent cells delayed development of cancer.

Mixed group dying of cancer lived 25% longer. 


Survival non-cancer death increased 30%.

Xu (2018).Increased post treatment survival 36%. Causes of death same. Leading cause death mice is cancer; therefore delayed death from cancer, 

6. Dasatinib, Zithromycin, Fisetin: The senolytic Cocktail

Four major senescent cell types

The three senolytics we use use are:

Dasatinib

Zithromycin

Fisetin.

Quercetin is very similar to Fisetin; but Fisetin is better. 


The four major senescent cells types and the drug used to target them are:

Adipocytes: Dasatinib

Fibroblasts: Zithromycin

Endothelial cells: Fisetin

Mesenchymal stem cells: Rapamycin (no effective senolytic available)

Adipocytes

H

Fibroblasts

"Azithromycin and Roxithromycin define a new family of "senolytic" drugs that target senescent human fibroblasts", Ozsvari, Lisanti; 2018. 


In 2018, a major study defined a new family of senolytic drugs. Azithromycin is the widely used antibiotic called Zithromycin and commonly prescribed as "Z-pak" a broad spectrum antibiotic. Roxthromycin is not approved in USA. Interestingly, the closely related parent drug, Erythromycin,  did not show senolytic activity. 


In this study they screened a large number of FDA approved antibiotics and these two drugs emerged. The two drugs kill senescent fibroblasts; but not normal fibroblasts. The senolytic effects was demonstrated against human lung fibroblasts and human skin fibroblasts. 


"Azthromycin preferentially targets senescent (fibroblasts) cells, removing approximately 97% of them with great efficiency. This represents a near 25-fold reduction in senescent cells."


They tested many other compounds. Notably the following compounds were ineffective:

Dasatinib

Quercetin

Rapamycin

Erythromycin

Doxycycline.


It turns out that Zithromycin has been used widely used as a senolytic to extend the life span and health for many years in patients with cystic fibrosis. Zithromycin is also used in other chronic lung  diseases with good effect. However, prior to this 2018 study it was never fully understood that Zithromycin was acting as a senolytic.


None of the prior papers on senescent cells and senolytics discuss Zithromycin. However, the use of Zithromycin to remove senescent fibroblasts appears to be a major addition to the family of senolytics drugs. Zithromycin and its use will be discussed in detail in following section.

Endothelial Cells

R

Mesenchymal stromal cells

H

C.

7. Azithromycin

Introduction

aa

why an antibiotic is a senolytic

B

cysric fibrosis

C

chronic lung diseas

D

skin disease

E

potential

fibrotic diseases

F

how use clinically

8. Treatment with Senolytics

Diseases Ameliorated by Removing Senescent Cells

  1. Aging                                                                         
  2. Cancer                                                                       
  3. Cardiovascular disease, Cardiomyopathy                                                                                             
  4. Alzheimer's Disease, Neurodegeneration                                                                                                
  5. Chronic Lung disease, Emphysema, Idiopathic Pulmonary Fibrosis
  6. Chronic Kidney disease, Glomerulosclerosis
  7. Non Alcoholic Fatty Liver
  8. Obesity, Overweight, Metabolic Syndrome
  9. Osteoarthritis, Osteoporosis
  10. Lumbar disc degeneration
  11. Eye: cataracts
  12. Muscle frailty
  13. Stem Cell Failure
  14. Death from COVID-19 (suspected)


In general, any age-related phenotype, a condition or disease which gets worse with age and has increased incidence with age, is likely a senescent cell related condition and is ameliorated with senolytics. This includes everything from COVID-19 to Alzheimer's disease.

Dasatinib

Dasatinib is the only senolytic clinically available for which there is extensive experience both in use with humans and animal trials. Dasatinib is sold under the brand name, Sprycel. It was developed by Bristol-Myers Squibb and approved for medical use in United States in 2006. It is used to target certain cases of chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). It is taken by mouth. Elimination half is 1.3 to 5 hours. it is a tyrosine-kinase inhibitor. 


Dosage: 100 mg/day. Leukemia: every day for years; sometimes taken 4 days on, 3 days off to reduce side effects.


Side effects: 

Mild to moderate diarrhea, peripheral edema, headache. 

Others: low white blood cell count, low blood platelets, anemia.

Severe side effects: bleeding, pulmonary edema, heart failure, pleural effusion, pulmonary hypertension. 


List price:  $9,353 for # 30, 50 mg tablets.

GoodRx coupon: CVS $8,362.


My office: $10 for a 50 mg capsule, Dasatinib.

Dasatinib is bought from Kentucky compounding pharmacy and shipped to patient. 


Usual dose: 2 tabs a day for 3 days, cost for usual course treatment: $60.00.

Senolytic Treatment in Clinical Practice

Dasatinib is my preferred senolytic drug due to excellent results in animal, small human trials and 15 years clinical experience. It is the most studied approved prescription drug which has been demonstrated that it can be repurposed for off label use as a senolytic.


There are 3 human clinical studies discussed in section 2 using Dasatinib as senolytic. 


Dasatinib stuudies include 10 mouse studies discussed above:

1 Mouse life span and health

2 Heart, cardiovascular

2 Alzheimer's disease

1 Pulmonary fibrosis

1 Fatty Liver

1 Osteoporosis

2 Metabolic syndrome


The Senolytic Cocktail: Dasatinib + Quercetin +Fisetin

Dasatinib is the drug of choice as there are three human clinical studies, excellent results in animal trials, and it is well tolerated.


Quercetin: Used in two of three human studies. All mouse studies with Dasatinib included Quercetin.  Effectiveness used alone not shown; but very safe and no serous reported side effects. Included in formula: 1000mg dose.


Fisetin had excellent result in 2018 study (Yousefzadeh) discussed above. Fisetin is very similar to Quercetin, but more effective. The main problem with Fisetin is limited data. Only one mouse study and there are no human studies published. (Fisetin is a naturally occurring substance sold on internet without patent and there is no commercial interest in promoting Fisetin.) Fisetin is very safe and well tolerated.  Due to limited experience with Fisetin I do not use Fisetin as a stand alone treatment; but include it with Dasatinib as the better Flavinoid.


My method is use all three:


Dasatinib 100 mg dose for 3 days.

Quercetin 1000 mg for 3 days

Fisetin 1500 mg for 3 days.


Dasatinib requires a prescription.

Quercetin sold as supplement internet 500 mg tabs. 

Fisetin  sold  as supplement on internet as 100 mg and 250 mg tabs.


The above is considered as the maximum dose. Patients can begin with a smaller dose it determine sensitivity.

Dasatinib and Senolytic Clinical Treatment Protocol

1-2 treatments a month (3 day course 100 mg Dasatinib each day): 

for serious disease such as Alzheimer's disease, cardiomyopathy, ASHD, pulmonary fibrosis, emphysema, fatty liver, chronic kidney disease, metabolic syndrome,  obesity, frailty.


4 treatments a year  (3 month interval):

for lifespan, health span.


Best age to start: 40-45 years.


For anti-aging, Baker (2016) showed best results when started in 12 month old mice (40 years human). Median life span extension was 35%. This would suggest middle-age in humans or about 40-50 years  as the best age to start. 


However, in Xu, D+Q was started in very old mice which were said to be the human equivalent  of 75-90 years. There was a reported 36% increase in lifespan calculated from time D+Q was started. The Xu study showed senolytic could still be beneficial in the elderly.


As regards various disease, treatment is always best for pre-pre-disease. The first step in many age-related disease is the accumulation of senescent cells. The best treatment is early removal of senescent cells before clinical disease develops. An example of this is treating insulin resistance BEFORE the development of overweight/obesity. 


However, removal of senescent cells can also be helpful to ameliorate established disease. In established clinical disease, accumulation of large numbers of senescent cells is may be driving acceleration of disease progression. Both disease progression and number of senescent cells both tend to follow a exponential progression. 


HIT and RUN:

It takes senescent cells time to accumulate. The time frame may be a few weeks for senescent cells to form. Intervals used in various mouse studies have been twice a week, weekly, once every 2 weeks, once a month as intervals in various studies. 


In the case of Dasatinib, interval dosing and not daily dosing is used to avoid side-effects. 


The best dose to use at this point in time is empirical. It is based upon experience and medical judgment in the absence of extensive published clinical studies in humans regarding treatment of various diseases. Dose and interval must be extrapolated from mouse studies, which is very imprecise.


Empirical dosing schedules are not optimal.


You go to war with what you have; not what you want. This is what we have at this point in time. 


Combined use of Rapamycin plus Dasatinib

There are no published studies involving combined use of rapamycin plus senolytics. I expect the combined use will have excellent effects on both aging and age-related disease. They both attack senescent cell diseases. Rapamycin decreases production of senescent cells and rapamycin opposes the action of SASP. Senolytics remove senescent cells. Using Alzheimer's disease as an example. Studies  suggest rapamycin may prevent Alzheimer's disease and senolytics may ameliorate established disease. 


The use of senolytics for treatment of aging and age-related disease is based primarily on animal studies. There are no large human clinical studies. This is a work in progress. 

9. zithromycin Doxycycline Vitamin C rx Cancer Stem Cells

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10 Doxycycline

Doxycycline's many effects

In Blagosklonny's classic paper, "From Rapalogs to anti-aging formula," 2017; Doxycycline is included in the anti-aging forumula: 

Doxycycline:


"Doxycycline, a broad-spectrum antibiotic of the tetracycline class, extends lifespan in C elegans [220] and Drosophilia [221, 222]. Doxycycline suppresses tumor growth and metastasis in mice [223. 224]. Importantly, doxycycline is a component of an anti-metastatic combination, which includes doxycycline, aspirin, lysine and mifepristone [225]."


The tetracyclines and Doxycycline have many actions. Above Blagosklonny noted two classes of actions, anti-aging  and anti-cancer as reasons for inclusion in anti-aging formula. The anti-cancer in particular is extremely important. That is especially true because Doxycycline as part of combination with Zithromycin and Vitamin C can be used to target cancer stem cells.


The actions of Doxycycline include:

1. Antibacterial

2. Life span

3. Anti-Cancer

4. Anti-Amyloid:  Alzheimer's Disease

5. Anti-Amyloid: Parkinson's Disease

6. Inhibition of MMP

7. Scavenging Reactive Oxygen Species

8. Anti-Inflammatory Effects


Doxycline and Life Span

Doxycycline use as antibiotic is because targets bacterial ribosomes. Bacterial ribosomes are very similar to mitochondrial ribosomes which is basis to target the mitochondrial ribosomes of cancer stem cell. 


Ref 220: "Mitonuclear protein imbalance as a conserved longevity mechanism", Houtkooper, 2013. 

AG: Very complex study. About how using mitochondria to change life span up or down. Doxycycycline used to target mitochondria. changed lifespan in both mice and C. elegans.


They note that rapamycin targets nuclear DNA; but doxycycline targets mitochondria proteins.


 



Doxycycline and Cancer

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Doxycycline and Alzheimer's Disease

 "Doxycycline for Alzheimer's Disease: Fighting B-Amyloid Oligomers and Neuroinflammation", Balducci , Milan, 2019.

Doxycycline and Parkinson's Disease

"Repurposing doxycycline for synucleinopathies: remodelling of  a-synuclein oligomers towards non-toxic parallel beta-sheet structure species:; Gonzalez-Lizarraga, 2017.

Summary

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Inhibition of Matrix Metalloproteinases (MMP)

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Anti-Inflammatory effects

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11. Vitamin C and Anti-Aging

Introduction Vitamin C

The combination of two antibiotics Zithromycin and Doxycycline combined with simultaneous us of Vitamin C is proposed above as a novel treatment of cancer stem cells. Prior to this Vitamin has a long history of use as solo agent. 

Vitamin C serum levels and Mortality and Disease

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12. The new Anti-Aging Forumula

Rapamycin

Dasatinib

Fisetin

Zithromycin

Doxycycline

Vitamin C

Metformin



Dose and Interval

Rapamycin 3-6 mg; once a week


Dasatinib 50 mg capsules;  2 capsules a day for 3 days; once a month to 4 times a year.


Fisetin 100 mg capsules; 1500 mg once a day for 3 days; once a month to 4 times a year; taken with Dasatinib.

[Quercetin 1000 mg can be use in addition to Fisetin]


Zithromycin 250 mg tablets; 1 tab day for 6 days; once a month. 


Doxycycline 50 mg capsules, 1-2 capsules a day.


Vitamin C capsules; 250-500 mg a day.


Metformin ER, 500 mg 1-4 tabs a day. 


Other valuable drugs:

Statins

ARBs (candesartan)

Tadalafil

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