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Is there a known glucosepane cross-link breaker?

Is there a known glucosepane cross-link breaker?


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I read the following on wikipedia:

There is, however, no agent known that can break down the most common AGE, glucosepane, which appears 10 to 1,000 times more common in human tissue than any other cross-linking AGE.

(AGEs are related to oxidative stress, aging and chronic diseases.)

I was wondering maybe the article is out of date, and there is a glucosepane cross-link breaker available. Do you know anything of that, or a current research which aims to find that?


Unfortunately at this time, there are no known glucosepane breakers which can feasibly be used in a living organism. There is a small (very small) amount of research being done on the topic by Yale university in cooperation with the SENS foundation, but the focus is extremely early stage - nothing more than synthesis and detection of artifical glucosepane and precursors.

It is a rather frustrating situation, as there are very good arguments indicating glucosepane as an almost universal 'bad thing that should be fixed', and the molecule itself isn't particularly complicated. It seems that this would be a low effort/high reward research area.


You may want to look at Chebulic Acid as a potential inhibitor of AGE cross linking AND a breaker of AGE cross-linking.

Review this article: http://biofoundations.org/?p=1004


The problem is the glucosepane bond is very strong, hence the molecules which would break this bond need be necessarily toxic and damaging to other molecules in the body. Glucosepane breakers already exist, but they are too toxic, and perhaps all will necessarily be that way. All hope is not lost however. Other very effective medical drugs (like clot breaking agents) are extremely toxic although life saving if applied in a local fashion via angiography under controlled conditions. My hunch is that the same will need to be done for the known glucosepane breakers. Glucosepane is extracellular, so the problem is really to avoid liver and renal toxicity. Animal studies should concentrate on delivering known glucosepane breakers under special conditions of renal, liver, and cardiovascular and respiratory support and though hyper-hydration, hypothermia, ECMO, dialysis, metabolic blocking agents, etc, to limit systemic toxicities. It won't be easy, but it could be well worth the effort if such single shot therapies could be developed which might then have long lasting effects as glucosepane would take many years to decades to build up again.


I came across this new research from Yale University:

We are currently in the process of evaluating the enzyme's mechanisms of action and identifying other metabolites generated. This is the first demonstration that glucosepane can be broken down enzymatically.

http://diabetes.diabetesjournals.org/content/67/Supplement_1/1229-P


Is there a known glucosepane cross-link breaker? - Biology

Glycation and crosslinking have been implicated as strong contributors to many progressive diseases of aging, including vascular diseases (such as atherosclerosis, systolic hypertension, pulmonary hypertension, and poor capillary circulation), erectile dysfunction [Usta], kidney disease, stiffness of joints and skin, arthritis [deGroot, Verzijl], cataracts, retinopathy, neuropathy, Alzheimer's Dementia [Ulrich, Castellani], impaired wound healing, urinary incontinence, complications of diabetes, and cardiomyopathies (such as diastolic dysfunction, left ventricular hypertrophy, and congestive heart failure). [Bucala]

Arterial stiffening causes an increase in the pulse pressure wave which travels through the blood vessels with each beat of the heart. Pulse pressure is measured by subtracting diastolic blood pressure (low number) from systolic blood pressure (high number).
( P = S - D ).
(For example, a patient with blood pressure of 155/90 would have a pulse pressure of 65.) Increased pulse pressure due to arterial stiffening is a leading risk factor for cardiovascular disease and brain stroke in the elderly. [Kass]

Diabetic Complications

Inhibitors of Glycation Crosslinking

Crosslink Breakers

Clinical Trials

So far, the safety profile of the drug appears to be excellent in human subjects. However, in December 2004, a study feeding alagebrium to lab rats for two years, found an increased rate of liver cell alterations in male rats, but not the females. This strain of lab rat (Sprague-Dawley) often develops liver cell alterations spontaneously, without drugs. The increased rate is comparable to effects caused in Sprague-Dawley rats by other, already approved drugs, such as the statins. These abnormalities are not necessarily expected in humans. Following further study, FDA allowed clinical trials to proceed.

No harmful interactions with other drugs have been observed. Any subjects who had been already taking other blood pressure medications continued on their previous medications in addition to taking alagebrium. Benefits of alagebrium were observed in addition to any benefits from the other blood pressure medications.


Dealing with Glucosepane

Advanced glycation end products (AGEs) represent a family of protein, peptide, amino acid, nucleic acid and lipid adducts formed by the reaction of carbonyl compounds derived directly or indirectly from glucose, ascorbic acid and other metabolites such as methylglyoxal.

The Amercian Chemical Society has this to say about AGEs:

In biological systems, some proteins react with open-chain carbohydrates to form adducts that contain amino acid and carbohydrate residues. When the carbohydrate is d-glucose, the product is glucosepane, which has lysine and arginine components.

Glucosepane belongs to a group of substances known as advanced glycation end products, or AGEs. It is by far the most common AGE in human tissue.

The website LEAF Science discusses glucosepane in relation to aging as follows:

A suspected reason we age is the accumulation of sugary metabolic wastes known as advanced glycation end-products (AGEs). AGEs are wastes that are, in some cases, hard for our metabolism to break down quickly enough or even at all.

There are various kinds of AGEs present in the body, although none are as common as glucosepane, which is the most abundant by a huge margin. Glucosepane is very hard for the body to break down (if indeed it can at all), and it can last several decades once formed.

AGEs, such as glucosepane, form cross-links, binding together important proteins such as those making up the supporting extracellular matrix scaffold and preventing them from moving.

The elastic properties of skin and blood vessel walls are due to the extracellular matrix having a particular structure, and cross-links degrade that structure, preventing it from functioning correctly. The presence of AGEs contributes to blood vessel stiffening with age and is implicated in hypertension and diabetes[1-2].

So it seems clear that removing or inhibiting glucosepane formation could have an impact on slowing or reversing aging. Wikipedia describes three methods being investigated to do this:

Α-Dicarbonyl trap [ edit ]

One method attempted to inhibit glucosepane formation is to use an α-dicarbonyl trap molecule, aminoguanidine (AG). AG reacts with the α-dicarbonyl intermediate with a higher affinity than arginine, thus blocking the cross-link. While this method has been seen to have some success, it did not greatly interfere with the normal aging of rats. [26]

Thiazolium salts [ edit ]

Another method that has been investigated is the use of thiazolium salts to break the a-dicarbonyl intermediate, therefore cutting off the reaction pathway that leads to glucosepane. These compounds are thought to act as bidentate nucleophiles that attack the adjacent carbonyls in the alpha-dicarbonyl intermediate, which then leads to the cleaving of the C-C bond between the carbonyls. [27] However, an alternate hypothesis as to how they work is that they act as chelating agents. [28] Two thiazolium molecules, PTB (N-phenacylthiazolium bromide) [29] and ALT-711, [30] have demonstrated success at reducing glucosepane levels in rats.

ECM [Extra Cellular Matrix] turnover [ edit ]

A completely different approach to reducing cross-links that has been proposed is enhancing the ECM turnover processes, which would force the degradation of cross-linked proteins to replace them with new. However, a potential downside to this would be leaky blood vessels resulting from too far enhanced turnover. [31]

Of the three, only the last actually aims to remove glucosepane from the system. There are two proposed routes to doing this, one is through chemical agents and the other through biological agents. The SENS Research Foundation proposes both as follows:

ECM Turnover Stimulation would activate or enhance natural processes to digest old ECM and replace it with new. It will be important to tune the collagen degradation to a rate slow enough to prevent dire side effects, such as hemorrhage from leaky blood vessels as collagen molecules are removed and replaced. Glucosepane Breaker Discovery would use rational drug design and high-throughput screening to find small molecules which are able to break glucosepane crosslinks of extracellular proteins. Candidates would be further screened for selectivity in order to ensure that only glucosepane gets broken.

As far as inhibiting glucosepane formation the best studied compound is metformin:

Metformin is the current treatment of choice for type 2 diabetes due to its glucose lowering effect [ 134 ]. MG [dicarbonyls methylglyoxal] lowering effects of metformin have been reported in addition [ 158 ]. The mechanism behind the decrease in MG levels by metformin is uncertain. Metformin reacts with MG via its guanidine group but the reaction proceeds slowly [ 159 ]. Nevertheless, a MG-metformin adduct has been detected in urine of patients treated with the drug at concentrations up to 4.3 μM [ 156 ]. Lowering of MG levels by metformin is associated with an increase of GLO1 [glyoxalase 1] in peripheral blood mononuclear cells and a trend for increase in red blood cells [ 160 ]. Thus metformin may inhibit AGE formation directly through lowering of MG levels via two ways, namely MG scavenging and induction of GLO1 activity.

Anti-diabetic plants and spices have also been the subject of initial study here:

High molecular weight protein products representing the dimer, trimer and tetramer of lysozyme were detected in the presence of fructose. Among the nine antidiabetic plants, seven showed glycation induced protein cross-linking inhibitory effects namely Ficus racemosa (FR) stem bark, Gymnema sylvestre (GS) leaves , Musa paradisiaca (MP) yam, Phyllanthus debilis (PD) whole plant, Phyllanthus emblica (PE) fruit, Pterocarpus marsupium (PM) latex and Tinospora cordifolia (TC) leaves . Inhibition observed with Coccinia grandis (CG) leaves and Strychnos potatorum (SP) seeds were much low. Leaves of Gymnema lactiferum (GL), the plant without known antidiabetic effects showed the lowest inhibition. All three spices namely Coriandrum sativum (CS) seeds, Cinnamomum zeylanicum (CZ) bark and Syzygium aromaticum (SA) flower buds showed cross-link inhibitory effects with higher effects in CS and SA. PD, PE, PM, CS and SA showed almost complete inhibition on the formation of cross-linking with 25 μg/ml extracts.

Further work on polyphenols and flavanols is carried out here:

Physiological concentration of Mg 2+ , Cu 2+ , and Zn 2+ accelerated AGE formation only in glucose-mediated conditions, which was effectively inhibited by chelating ligands. Only quercetin (10) inhibited MGO-mediated AGE formation as well as glucose- and ribose-mediated AGE formation among 10 polyphenols ( 1–10 ) tested. We performed an additional structure-activity relationship (SAR) study on flavanols ( 10 , 11 , 12 , 13 , and 14 ). Morin ( 12 ) and kaempherol ( 14 ) showed inhibitory activity against MGO-mediated AGE formation, whereas rutin ( 11 ) and fisetin ( 13 ) did not. These observations indicate that 3,5,7,4′-tetrahydroxy and 4-keto groups of 10 are important to yield newly revised mono-MGO adducts ( 16 and 17 ) and di-MGO adduct ( 18 ) having cyclic hemiacetals, while 3′-hydroxy group is not essential. We propose here a comprehensive inhibitory mechanism of 10 against AGE formation including chelation effect, trapping of MGO, and trapping of reactive oxygen species (ROS), which leads to oxidative degradation of 18 to 3,4-dihydroxybenzoic acid ( 15 ) and other fragments.

With regards to actually breaking glucosepane down so that it can be removed, the only candidate compound proposed to date is rosmarinic acid. The following from Wikipedia:

Rosmarinic acid is a potential anxiolytic as it acts as a GABA transaminase inhibitor, more specifically on 4-aminobutyrate transaminase. [11] Rosmarinic acid also inhibits the expression of indoleamine 2,3-dioxygenase via its cyclooxygenase-inhibiting properties. [12]

Senescent leaves of Heliotropium foertherianum (Boraginaceae) also known as octopus bush, a plant used in many Pacific islands as a traditional medicine to treat ciguatera fish poisoning, contain rosmarinic acid and derivatives. [13] Rosmarinic acid may remove the ciguatoxins from their sites of action. [ citation needed ]

The use of rosmarinic acid is effective in a mouse model of Japanese encephalitis. [14]

There is much more we could say about this topic, and much more detail needed to fully understand the contents of this post. We shall endeavour to cover this in future posts. Thank you.


Abstract

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch’s membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.


Interview with David Spiegel (Glucosepane Synthesis)

CRISPR stole all the headlines last year. A possibly more substantial advance (for near-term rejuvenation therapy) went under the radar. Spiegel Labs synthesized glucosepane. Better understanding of how glucosepane is created and having the molecule readily available for research could rapidly advance anti-aging treatments as glucosepane is one of the key indigestible extracellular cross-link molecules that accumulates as humans age and causes stiffening and malfunction of many tissues.

Coming up on the LongeCity Now podcast on February 11th, is PhD, MD David Spiegel, head of Spiegel Labs.

Put your chemistry hats on and submit questions ahead of time in this forum thread.

Attached Files

Edited by Mind, 05 February 2016 - 10:45 PM.

#2 Antonio2014

Thanks for contacting him, Mind. Here my questions go:

- What's next for glucosepane research in your lab? What are you working on now?

- Regarding breaking of glucosepane crosslinks, some people think that crosslink-breaking enzimes will be too big to reach the links that must be cut in collagen fibrils and thus prefer small molecules. Other people think that small molecules will be too unspecific for the task. What do you think? What's your preferred strategy for the search of a glucosepane crosslink breaker?

- Is your glucosepane synthesis process patented? Did you contact a company to start production of glucosepane for other researchers?

- Why did you enter the glucosepane/AGE research field? It was because you were interested in aging or diabetes? Did you know about SENS at the time?

- How far away are we from the first efficient glucosepane-detecting antibodies?

Edited by Antonio2014, 06 February 2016 - 10:33 AM.

#3 Mind

Great questions Antonio, thanks.

#4 Antonio2014

One more: Do you think that molecular dynamics simulations could be useful for searching for glucosepane breakers? (Maybe here at Longecity we could coordinate such an effort, through BOINC or whatever.)

#5 YOLF

How soon will we have a supplement that is effective against Glucosepane? A stronger more effective pharmaceutical?

#6 niner

One more: Do you think that molecular dynamics simulations could be useful for searching for glucosepane breakers? (Maybe here at Longecity we could coordinate such an effort, through BOINC or whatever.)

There are (minimally) two aspects to designing a glucosepane breaker. One is molecular recognition of glucosepane and the other is engineering the cleavage of bonds somewhere on the molecule. There are a lot of computational tools for finding small molecules that might bind to the concavities of a particular macromolecule, but glucosepane turns that problem inside-out. There's not a pocket to bind a small molecule in, so you'll probably need something that "engulfs" or wraps around the glucosepane linkage. Maybe there's a role for fragment-based drug design tools there, although it's kind of a long shot, imho. Essentially you need a catalytic antibody. (Such things exist.) This is a very unusual problem that would almost never come up in the traditional pharmaceutical industry, and that is the field that has driven computational drug design. Of all the computational tools that exist, most, including MD, are probably not going to be a great fit to this problem. I'd broaden Antonio's question to: "Do you see a role for any computational tools in the design of a glucosepane breaker?"

And one more: Will there be any structural characterization (for example, x-ray crystallography) of a glucosepane linkage?

#7 Mind

Thanks for the question niner. I was able to get some of that type of information from the interview (which was yesterday). The Spiegel group is thinking about those issues. Should have the podcast available next week.

#8 Mind

File Name: LongeCityNow_David_Spiegel2016.mp3

File Submitter: Mind

File Submitted: 18 Feb 2016

File Category: Podcasts

Guest: David Spiegel

Find out about the latest research into glucosepane and other activities at the Spiegel Research group.

#9 Mind

Thanks for all the great questions for this interview. I think you will find a good level of detail in the answers provided by Dr. Spiegel. He explains quite well the next steps in finding a glucosepane breaker.

#10 Antonio2014

Mmm. I can't download the file.

Not Found

The requested URL /media/LongeCityNow_David_Spiegel2016.mp3 was not found on this server.

Additionally, a 404 Not Found error was encountered while trying to use an ErrorDocument to handle the request.

Apache/2.2.27 (Unix) mod_ssl/2.2.27 OpenSSL/0.9.8e-fips-rhel5 mod_jk/1.2.37 mod_bwlimited/1.4 PHP/5.4.30 Server at www.longecity.org Port 80

#11 corb

Yup. Same for me. I get a 404.

#12 Avatar of Horus

You need to add another ".mp3" into the link:

  • Informative x 1
  • Agree x 1

#13 corb

Nice interview.
If they ever get around to making a [email protected] I'll consider investing in couple of 2nd hand workstation pcs to crunch that 24/7.

#14 Antonio2014

Very interesting interview. I too would donate the time of my computers that are now crunching for BOINC.

Edited by Antonio2014, 19 February 2016 - 11:13 AM.

#15 ceridwen

#16 Mind

Arrgg. 404 is the bane of my existence.

#17 reason

The Longecity community leadership runs a regular podcast series, interviewing notable advocates and researchers in the longevity science community. The latest podcast is a discussion with researcher David Spiegel at Yale on the topic of glucosepane cross-link breaking. His research group, funded in part by the SENS Research Foundation, is working towards the means to remove glucosepane cross-link accumulation as a contributing cause of aging. Loss of tissue elasticity lies at the root of arterial stiffening, hypertension, and cardiovascular disease, for example, but this is only one of many problems caused by the growing numbers of persistent cross-links in old tissues. You can look back in the Fight Aging! archives for a long post from earlier this year that outlines the present state of research in this field, so I won't cover the same ground here, but rather skip straight to the podcast transcript:

Justin Loew: Welcome back to Longecity Now. Some of you have been following the SENS theory of aging for over a decade now, and might be wondering if there is any progress. The answer is "yes", as we learned from a podcast with Aubrey de Grey late last year. In that interview Aubrey mentioned the artificial synthesis of glucosepane had recently been achieved. This is important because glucosepane is suspected be a significant culprit in aging tissues. In this edition we hear from the head of the lab that artificially created glucosepane. For those of you who are dying to hear more of the technical details of aging interventions, this interview with David Spiegel should satisfy your curiosity.

David Spiegel: Hello! Great to be here.

Justin Loew: As a little background, how did you come to be interested in synthetic chemistry? Was it mostly scientific curiosity, or was it a determination to cure human diseases?

David Spiegel: So, it's funny, I often get asked this question. I was probably a six-year-old kid, asked in second grade what I thought I would be doing in the year 2000, at the time still 21 years away. I still have the document in which I wrote that I wanted to be a chemist in a drug company. And so, I have stayed pretty true to that vision for my life. I have always been fascinated by molecules, and the fact that simple chemical matter has profound changes on human beings. So chemistry was a natural outgrowth of that interest, and in particular the idea that I could rationally design drugs to do things that nobody else had thought a drug could do. So that has led to research interests in my lab, one of which is in the area of immunotherapeutics, new kinds of molecules that can manipulate the immune system, to do interesting and cool things there. Also, the idea that drugs, small molecules, can be useful in reversal of the aging process.

Justin Loew: Your synthetic chemistry lab made headlines last year for synthesizing glucosepane. Many listeners are familiar with the theory that glucosepane is possibly a significant contributer to the aging process, being an extracellular cross-linking molecule that stiffens tissues, but most less familiar with the reasons why it is so difficult to do anything about it. Why has science been so stymied in regards to this molecule, even though it has been known for decades.

David Spiegel: Yes, it is a good question. So, it is a very difficult molecule to make. Well, two issues: first it is very difficult molecule to make, but also it is actually a difficult molecule to isolate. So even though it is found in all of us, it is found in our tissues, our bones, trying to isolate it in a pure form from the human body is incredibly difficult. Only very small quantities are obtained, and the compounds isolated are actually mixtures of very similar stereoisomers, a kind of different versions of glucosepane that simply can't be separated. So from my perspective I thought it would be quite valuable to take on this challenge, and that is really one of the main areas of focus for my laboratory, which is making very difficult molecules using techniques in organic chemistry. So in my mind, this is something that believed in for a long time. For glucosepane, it is a perfect marriage of interesting chemistry and incredibly interesting biology. The biology here is hard, and people have had a hard time, as you said, studying glucosepane, and of course making it has proven an incredibly difficult challenge because of its complex and intricate chemical structure. So we've been very interested in making it, and now we're in the phase of seeing what we can do with it, particularly with the goal of breaking glucosepane, or developing agents that can break glucosepane, that we think can actually reverse the pathology associated with aging.

Justin Loew: And on that, to add to the pathology aging, do you have any idea on how big of a role glucosepane plays in the aging process?

David Spiegel: You know, there is certainly a lot of evidence indicating that glucosepane levels correlate with organ damage and diseases like diabetes, and there is an argument that in diabetes one of the hallmark features is a kind of accelerated aging of the tissues. Also in people who are simply older, in people greater than 65 years of age, it turns out that there is more glucosepane found in collage than there are enzyme-catalyzed cross-links, the cross-links that are actually supposed to be there are outnumbered by glucosepane. It is these very tissues that are involved in the disease of old age. So collagen-containing tissues include blood vessels, bones, joints, and what do we see in old age? We see cardiovascular disease, we see joint disease, we see renal disease, often. So there is a lot of correlative evidence that is backed by with reasonable mechanistic speculation about a causative role that glucosepane can play, that I think really does implicate it as a key factor in what we term the pathophysiology, the damage, the disease, the element of old age that is a disease.

Justin Loew: Now that you made the molecule, and are looking at breaking the molecule, do you have any estimate of how long it might be before there is an effective therapy that addresses glucosepane?

David Spiegel: That's a good question. I think that from the standpoint of basic research, we've already made some progress in identifying some potential strategies for breaking glucosepane. As you know, there is a significant regulatory challenge associated with bringing new therapeutics to market, and so if I had to estimate - well, this is a very high bar in terms of . well it is an extraordinary challenge, just the idea of making therapeutics that can break a molecule is kind of an untested concept. But the progress we are making, and the surge of interest right now in protein and enzyme-based therapeutics in pharma, makes me speculate that it is possible we could have something that is therapeutically viable on the order of 10-20 years from now. That may not seem like a short time, but from a therapeutics perspective, I think it is within our kind of vision.

Justin Loew: Staying on that kind of thought there, that the breaking of glucosepane cross-links could be very important for aging research, some people think that cross-link breaking enzymes would be too big to reach the links that must be cut in collagen fibrils, and prefer small molecules. Other people think that small molecules would not be specific enough for the task, what do you think? What is your prefered strategy?

David Spiegel: That's another excellent question. I think that as a small molecule chemist, I would love nothing more than to develop a small molecule that could break glucosepane cross-links, and it is certainly something we've been thinking about for quite some time. I think it is actually a very difficult challenge for a small molecule to break a stable cross-link like glucosepane. Mechanistically speaking, in terms of the underlying chemistry, I think it's not clear how a small molecule would function. Now, on the enzyme side, or I should say on the protein side, I think it's possible to imagine low molecular weight enzymes that could be tissue-permeable to the extent that they actually do reach glucosepane cross-links. So my preferred strategy is a protein agent, but by all means I encourage anyone out there listening, and I'm also encouraging people in my own lab group, that small molecule strategies should not be abandoned. I think that both strategies are viable, but the one I see succeeding on the shortest time frame is probably an enzyme.

Justin Loew: Other work in your lab has revolved around using synthetic molecules to detect cancer, and encourage the immune system to attack. Do you think antibodies could be brought to bear against glucosepane?

David Spiegel: Absolutely, and I should say our lab is in the process, and we're making great strides towards identifying the first selective anti-glucosepane antibodies with just that goal in mind. One can imagine an antibody that can bind to glucosepane, and have attached to it some kind of catalyst that would enhance the breakdown of glucosepane. One could also imagine an antibody that is useful for the diagnosis, the detection of glucosepane cross-links in tissue, and so I think that antibody strategies are really high on the list.

Justin Loew: A lot people who would like to help out in this type of research but don't have the expertise use crowdsourced computing efforts such as [email protected] Could the search for a glucosepane breaker be helped by this type of work?

David Spiegel: Absolutely, and in fact we've certainly discussed those efforts. We have collaborators who have started work along those lines for computationally modelling the role of glucosepane in collagen cross-links, and with that information in hand, it really could be possible to develop a kind of hypothetical mechanistic strategy. When I say mechanistic I mean how would a molecule work, what would the chemistry have to look like for an antibody, a small molecule, some other kind of therapeutic modality, to break down glucosepane. It does have a very unique and suprisingly stable chemical structure. In fact, breaking down glucosepane is more than just causing it to degrade. One would also need to cleave the molecule in such a way as to separate the lysine and arginine strands that are being cross-linked by glucosepane, such as to restore the mobility and flexibility in the tissues that are being cross-linked.

Justin Loew: Then for the do-it-yourselfers who might be into synthetic chemistry, or for the other labs who might be listening in, is the molecule you synthesized patented? Is your university licensing the process or the molecule?

David Spiegel: Yes, so it is patented. We are in discussions surrounding licensing the molecule. We are also providing the molecule to the community for basically the cost it takes for us to make it. We want to encourage efforts of all kinds to find glucosepane breakers, so making it commercially available and developing collaborations with other laboratories are all very high on our priority list. For the do-it-yourselfers out there who are interested, feel free to contact me, and we can certainly make an arrangement where our lab will provide glucosepane for research purposes.

Justin Loew: They should just look online for the Spiegel Research Group at Yale University, and they'll be able to contact you or a member of your lab?

David Spiegel: Correct.

Justin Loew: Great! And lastly here, what other research is underway in your lab currently, something people should be keeping an eye out for?

David Spiegel: We have a number of research programs devoted to aging and age-related cross-links. I should also point out that we have been very grateful to the SENS Research Foundation for funding our work - Aubrey de Grey, William Bains, Michael Kope, and others at the organization have just been incredible in terms of the vision for funding this. This is fairly high risk research. We have antibodies, we are developing reagents for detecting a wide variety of advanced glycation end-products, all of which we believe are involved in the aging process. We also have a major effort, and as I mentioned before, in the development of new immunotherapies. So we're using small molecules that we designed to seek out various kinds disease-causing cells, organisms, proteins, for detection by the immune system. So we can actually make molecules that can alert the immune system to the presence of disease-causing factors that the immune system might have missed. So there is obvious therapeutic potential there, not only in aging, but also in cancer, infectious disease, autoimmune disease, and a whole range of other conditions as well.

Justin Loew: Well, that does sound very promising. We'll all look forward to future research publications from your lab. Dr. Spiegel, thank you for joining me.

David Spiegel: Thank you! Great to be a guest.

Justin Loew: It is refreshing to hear of the collaboration between SENS and the Spiegel research group. It seems that SENS has achieved good results from this investment. The problem is that the money is running out. Dr. Spiegel informed me that funding at his university is drying up, and Aubrey de Grey mentioned the same thing late last year in regard to SENS. This means that your support for rejuvenation research is even more crucial this year, as the world economy slows down. As a non-profit that advocates for life extension and provides funding for small-scale research, Longecity has the power to help out. Please consider joining us as a member, and watch for Longecity-approved fundraisers through 2016. Until next time.

As ever, progress in the field of rejuvenation research is constrained far more by lack of funding than by the difficulty of the challenges involved. The challenge in bootstrapping a movement is always the leap from funding source to funding source, the need to raise enough to get things done, and then build on that progress to attract the next source of revenue. Collectively we have achieved great success in the past fifteen years, going from no investment in SENS to tens of millions devoted to this field. That, of course, is just a set up for the latest leaps in search of more funding, enough to carry out the work that remains to be done. It is amazing the degree to which persuasion is required to get people to help in saving their own lives in the future, but that is the nature of the world we live in.


Building the Glucosepane Research Toolkit Continues with the Creation of Anti-Glucosepane Antibodies

Glucosepane is likely the most important form of persistent cross-linking in aging human tissue. There is some remaining uncertainty, but it appears that the vast majority of cross-links in old tissues are based on glucosepane. Cross-links are the consequence of advanced glycation end-products (AGEs), sugary metabolic waste that can bond with the structural molecules of the extracellular matrix. Where two such molecules are linked together by a single AGE (a “cross-link”), it reduces their ability to move relative to one another. The presence of many persistent cross-links thus degrades the structural properties of that tissue. This is particularly true of elasticity, vital to the correct function of skin and, more importantly, blood vessels. Cross-linking is likely an important contribution to arterial stiffening, and the hypertension and cardiovascular disease that follows as a consequence.

The solution to this aspect of aging is to find a way to periodically remove cross-links. That effort has been hampered by the fact that the important cross-links in humans and laboratory species such as mice are completely different. That was well demonstrated by the high profile failure of the cross-link breaker compound alagebrium to perform in humans in the same way that it does in rats. Further, the tools required to work with important human AGEs such as glucosepane have been lacking. Without necessary line items such as animal models, a cheap method of synthesizing glucosepane, and antibodies specific to glucosepane, scientists avoided this part of the field in favor of easier programs of research. Fortunately the SENS Research Foundation started to fund efforts to solve this tooling problem some years ago, and, once started and shown to be productive, that line of work has continued.

Today’s paper reports on the development of specific antibodies for glucosepane by the same group that first produced a robust, low-cost method of glucosepane synthesis. Antibodies that are highly specific to the molecules under study are needed for any rigorous program of development, as without them many assays of cells and tissues become questionable or impossible. This paper is an important step forward, just as much so as the synthesis of glucosepane. This part of the field of cross-link study is being opened up, and the more researchers to participate, the sooner we’ll see successful trials of cross-link breaking drugs capable of removing glucosepane from the human body. There is at present one startup biotech company working towards that goal, and in a better world there would be a dozen, a mirror of the developing senolytics industry.


Glucosepane is among the most abundant AGEs found in human tissues. It is formed from lysine, arginine, and glucose, and it is over an order of magnitude more abundant than any other AGE crosslink in extracellular matrix (ECM). Notably, glucosepane levels have been shown to correlate with various disease states, including diabetic retinopathy, microalbuminuria, and neuropathy. While the exact mechanisms behind glucosepane-mediated dysfunction remain unclear, it is believed to impair the functional and mechanical properties of proteins in the ECM and interfere with proteolytic degradation of collagen.

To date, the primary method for identifying glucosepane in tissues has required exhaustive enzymatic degradation followed by high pressure liquid chromatography-mass spectrometry (LC/MS). Although these protocols have proven effective in quantifying glucosepane in bulk tissue extracts, they are labor-intensive and the degradation process destroys the tissue architecture, making it difficult to examine the localization of glucosepane.

In recent years, anti-AGE antibodies have emerged as useful tools for studying AGEs and have the advantage of being compatible with the evaluation of intact tissues, enabling immunohistochemical staining and imaging procedures. Several anti-AGE antibodies have been produced by immunization of animals with AGEs generated either from total synthesis or through in vitro glycation methods. Such methods involve the incubation of an immunogenic carrier protein, such as BSA, with glucose or other reactive sugar metabolites. Reaction conditions that generate glucosepane are known also to generate a range of AGE by-products, including carboxymethyllysine. These in vitro preparation methods are unlikely to produce antibodies that are specific for glucosepane, although no such studies have been reported.

To avoid this expected complication, we decided to synthesize homogeneous, synthetic glucosepane immunogens. Herein, we describe the development and characterization of the first antibodies known to selectively recognize glucosepane. To this end, we have created a synthetic glucosepane immunogen that closely resembles glucosepane found in vivo and used it to generate a polyclonal antibody serum that recognizes glucosepane both in vitro and in ex vivo tissue samples. We have demonstrated that the antibodies can bind to glucosepane with high degrees of specificity and sensitivity through ELISA studies and have employed these antibodies in immunohistochemical experiments.

Interestingly, these latter studies demonstrate that glucosepane accumulates within sub-components of the retina, specifically the retinal pigment epithelium (RPE), Bruch’s membrane, and choroid, which are anatomic areas highly affected by AMD and diabetic retinopathy.


Conclusions and Clinical Significance

Treatment with an AGE cross-link breaker partially attenuated the alterations associated with cardiac function and SR Ca 2+ handling during diabetic cardiomyopathy. Since diabetic cardiomyopathy is a multifactorial disorder, these data suggest that AGE accumulation contributes to the impairment in excitation-contraction coupling by altering the function of SR Ca 2+ regulatory proteins, leading to a decreased ability for the diabetic myocardium to relax. Therefore, findings from this study provide novel mechanistic insights related to the pathogenic role of AGE accumulation on SR Ca 2+ handling in cardiac myocytes. Finally, since there is currently a lack of specific therapy to improve LV relaxation, findings from this study could have direct practical implications for the development of therapeutic strategies for patients with diabetic cardiomyopathy.


Methods

Six male, normotensive, nondiabetic rhesus monkeys (Macaca mulatta), aged 21 ± 3.6 years and weighing 8.6 ± 2.4 kg, were provided a 2-week adaptation period within the vivarium at the National Institutes of Health Primate Unit of the Poolesville Animal Center, an American Association for the Accreditation of Laboratory Animal Care-accredited center. Study protocols were approved by the Gerontology Research Center Animal Care Committee.

All cardiovascular evaluations were conducted in anesthetized animals without the use of endotracheal intubation, following standard protocols for sedation (Telazol, 3–5 mg per kg of body weight intramuscularly, after a 12-h fast). Systolic and diastolic arterial blood pressures (SBP and DBP) were recorded noninvasively with an automated sphygmomanometer cuff on the right upper extremity (Dinamap, Critikon, Tampa, FL). Mean arterial pressure (MBP) was estimated as (SBP − DBP) + DBP. Heart rate was recorded by using continuous electrocardiogram recordings. Three serial measurements of arterial stiffness indices (aortic PWV and carotid pressure pulse tracing) and two echocardiographic measurements of LV function were made during a 1-month period before drug administration (two animals underwent only one echocardiographic assessment at baseline). Subsequently, ALT-711 was administered once every other day over a 3-week period as 11 intramuscular injections, each 1.0 mg per kg of body weight. The study was of a single-arm, double-crossover (no drug, drug, no drug) design with baseline (predrug) measures as controls.

Arterial stiffness indices were measured again at 4, 6, 8, 11, 15, and 39 weeks after the last dose of ALT-711. Echocardiographic measurements were repeated at 4, 6, 11, and 39 weeks after the last dose of ALT-711. Arterial pressure waveforms were obtained from the right common carotid artery by applanation tonometry with a pencil-sized probe (Millar Instruments, Houston, TX) on the maximal pulsation of the artery as described (11). The carotid pressure pulse augmentation index (AGI) was determined from the average of 10 simultaneously recorded pressure waves by a custom-designed computer algorithm (12). As described (12), PWV was derived from simultaneous recordings of arterial flow waves from the right common carotid artery and the right femoral artery by using nondirectional transcutaneous Doppler flow probes (model 810A, 10 and 9 MHz, Parks Medical Electronics, Aloha, OR).

M-mode echocardiograms were obtained from two-dimensional guided images by using a 3.5-MHz transducer recording at 50 mm/sec. LV cavity end systole diameter (ESD), end diastole diameter (EDD), and septal and posterior wall thickness were measured with electronic calipers after the digitalization of individual frames in accordance with established guidelines (13). Several additional cardiovascular parameters were derived from the measurements of blood pressure and LV dimensions. LV fractional shortening (LVFS), a measure of myocardial contractility, and stroke volume index (SVindex) were estimated as follows: LVFS = (EDD − ESD)/EDD SVindex = EDD − ESD (the term SVindex refers to an estimate of stroke volume on the basis of a one-dimensional change in LV size between diastole and systole). End systolic pressure (ESP) was approximated as ESP = (2SBP + DBP). The effective arterial elastance (Eaindex = ESP/SVindex), a measure of total (i.e., both compliance and resistance components) LV vascular load (14–16), and ESP/ESD, a reflection of LV myocardial function, were calculated. Furthermore, ESD/SVindex, a measure of ventriculo-vascular coupling (5), cardiac output index (COindex) estimated as COindex = SVindex × heart rate (beats per minute), and total systemic vascular resistance index (TSRindex), derived from COindex and MBP, were calculated.

Arterial wave forms, pressure contours, and echocardiograms were evaluated by a single observer blinded with respect to the identity of the data. The reliability of waveform and pressure contour analyses was established by sequential measurements of the right carotid contour by the same examiner and reader on differing occasions in all monkeys. The repeated measurements were highly correlated (mean coefficient of variation 5%).

Statistical Analyses.

All results were expressed as mean ± the SEM, unless otherwise noted. Serial measurements were analyzed by using one-way ANOVA for repeated measures. The level of significance was set at P < 0.05, two-tailed.


Lalezari-Rahbar (LR) compounds

These are aromatic organic acids named after the developers, and are relatively new AGE inhibitors. Apart from anti-diabetic and anti-glycation effects, these have also been shown to have lipid-lowering benefits. Although several of their benefits are similar to those of aminoguanidine, these LR products are not carbonyl blockers. They are, however, good heavy metal chelators, just like carnosine and aminoguanidine. It is believed that even chelation alone, in the absence of carbonyl trapping, can be a good way of blocking AGEs.

Less useful anti-glycators (either because of a weak effect or because they are not easily available), are:

  • Penicillamine. Although not a potent anti-glycator, penicillamine can be used to improve arterial wall function which has been compromised due to glycation. In this respect, it has been used in association with vitamin E (19).
  • Tenilsetam (3-2-thienyl-2-piperazinone). This been used as a brain stimulant (nootropic). New research has examined its anti-AGE actions and its significant glycosylation-inhibiting benefits. It works like most cross-link blockers, namely by carbonyl trapping. In addition, Tenilsetam has antioxidant activities and copper chelating properties.
  • OPB-9195 (2-isopropyli-denehydrazono-4-oxo-thiazolidin-5-ylacetanilide). This carbonyl-trapping agent is a synthetic thiazolium derivative which inhibits cross-linking and improves kidney function. It also improves vascular function and reduces arterial thickening.
  • Pentoxifylline (brand name Trendal®) is normally used to improve circulation to the extremities.
  • Kinetin (furfuriladenine) brand name Kinerase®. In a study, kinetin inhibited carbonyl activity and reduced AGEs by up to 68%, but there are no new studies to confirm these benefits.
  • Kavalactones are some promising and relatively new inhibitors of protein glycation. These are also effective against lipid peroxidation (13).
  • Benfotiamine. This is a very promising compound which has several other actions as well as being anti-glycator. In a study, a combination of pyridoxamine and benfotiamine was found to improve pain, reduce inflammation and improve the quality of life in osteoarthritis patients (20).

Fresh Interview with Aubrey de Grey

Ariel Feinerman: Hello, Dr Aubrey de Grey!

Aubrey de Grey: Hello Ariel – thanks for the interview.

Ariel Feinerman: How do you feel 2018 year? Can you compare 2018 to 2017 or early years? What is changing?

Aubrey de Grey: 2018 was a fantastic year for rejuvenation biotechnology. The main thing that made it special was the explosive growth of the private-sector side of the field – the number of start up companies, the number of investors, and the scale of investment. Two companies, AgeX Therapeutics and Unity Biotechnology, went public with nine-digit valuations, and a bunch of others are not far behind. Of course this has only been possible because of all the great progress that has been made in the actual science, but one can never predict when that slow, steady progress will reach “critical mass”.

Ariel Feinerman: In 2017 SENS RF have received about $7 million. What has been accomplished in 2018?

Aubrey de Grey: We received almost all of that money right around the end of 2017, in the form of four cryptocurrency donations of $1 million or more, totalling about $6.5 million. We of course realised that this was a one-off windfall, so we didn’t spend it all at once! The main things we have done are to start a major new project at Albert Einstein College of Medicine, focused on stem cell therapy for Alzheimer’s, and to broaden our education initiative to include more senior people. See our website and newsletters for details.

Ariel Feinerman: What breakthroughs of 2018 can you name as the most important by your choice?

Aubrey de Grey: On the science side, well, regarding our funded work I guess I would choose our progress in getting mitochondrial genes to work when relocated to the nucleus. We published a groundbreaking progress report at the end of 2016, but to be honest I was not at all sure that we would be able to build quickly on it. I’m delighted to say that my caution was misplaced, and that we’ve continued to make great advances. The details will be submitted for publication very soon.

Ariel Feinerman: You say that many of rejuvenating therapies will work in clinical trials within five years. Giving that many of them are already working in clinical trials or even in clinic (like immunotherapies, cell and gene therapies) do you mean first – maybe incomplete – rejuvenation panel, when you speak on early 2020?

Aubrey de Grey: Yes, basically. SENS is a divide-and-conquer approach, so we can view it in three overlapping phases. The first phase is to get the basic concept accepted and moving. The second phase is to get the most challenging components moving. And the third phase is to combine the components. Phase 1 is pretty much done, as you say. Phase 2 is beginning, but it’s at an early stage. Phase 3 will probably not even properly begin for a few more years. That’s why I still think we only have about a 50% chance of getting to longevity escape velocity by 2035 or so.

Ariel Feinerman: Even now many investors are fearful of real regenerative medicine approach. For example pharmacological companies which use small molecules, like Unity Biotechnology, received more than $300 million, in much more favour than real bioengineering companies like Oisin Biotechnologies, received less than $4 million, even though biological approach much more powerful, cheap, effective and safe! Why in your opinion, and when can we see the shift?

Aubrey de Grey: I don’t see a problem there. The big change in mindset that was needed has already occurred: rejuvenation is a thing. It’s natural that small-molecule approaches to rejuvenation will lead the way, because that’s what pharma already knows how to do. Often, that approach will in due course be overtaken by more sophisticated approaches. Sometimes the small molecules will actually work well! It’s all good.

Ariel Feinerman: Do you agree, that small-molecule approach is generally wrong way in the future rejuvenation therapies? Because they have many flaws – especially their main mechanism via interference with human metabolism. Unlike them SENS bioengineering therapies are designed to be metabolically inert – because they just eliminate the key damage, they do not need to interfere with metabolism, so it is much easier than usual to avoid side effects and interactions with other therapies. They just eliminate the key damage, which means they are easier to develop and test – and much safer!

Aubrey de Grey: Ah, no, that’s too simplistic. It’s not true that small molecules always just “mess with metabolism” whereas genetic and enzymatic approaches eliminate damage. Small molecules that selectively kill senescent cells are absolutely an example of SENS-esque damage repair the only thing against them is that it may be more difficult to eliminate side-effects, but that’s not because of their mode of action, it’s because of an additional action.

Ariel Feinerman: In recent years many countries show green light for regenerative medicine. Fast-track approval in Japan, for example, which allows for emerging treatments to be used so long as they have been proven safe. The similar approach works in Russia. What about EU or USA?

Aubrey de Grey: There’s definitely a long way to go, but the regulatory situation in the West is moving in the right direction. The TAME trial has led the way in articulating an approvable endpoint for clinical trials that is ageing in all but name, and the WHO has found a very well-judged way to incorporate ageing into its classification.

Ariel Feinerman: Do you think of working with USA Army? As far as we know they make research on regeneration and are very interested in keeping soldiers healthier for longer. Then they have much money!

Aubrey de Grey: The Department of Defense in the USA has certainly funded a lot of high-impact regenerative medicine research for many years. I’m sure they will continue to do so.

Ariel Feinerman: Is any progress in the OncoSENS programme? Have you found any ALT genes? Is any ongoing research in WILT?

Aubrey de Grey: No – in the end that program was not successful enough to continue with, so we stopped it. There is now more interest in ALT in other labs than there was, though, so I’m hopeful that progress will be made. But also, one reason why I felt that it was OK to stop was that cancer immunotherapy is doing so well now. I think there is a significant chance that we won’t need WILT after all, because we will really truly defeat cancer using the immune system.

Ariel Feinerman: Spiegel Lab has recently published an abstract where they say they have found 3 enzymes capable of breaking glucosepane. Very exiting info! When can we hear more on their research? Revel LLC is very secretive company.

Aubrey de Grey: They aren’t really being secretive, they are just setting up.

Ariel Feinerman: When can we see the first human clinical trial of glucosepane breaker therapy?

Aubrey de Grey: I think two years is a reasonable estimate, but that’s a guess.

Ariel Feinerman: What do you think of the Open Source approach in rejuvenation biotechnology? Computer revolution in early 2000 has taken place only because Open Source caused an explosion in software engineering!

We have many examples when Big Pharma buys small company which has patents on technology and then cancel all research. In Open Source approach you cannot “close” any technology, while everyone can contribute, making protocol better and everyone can use that without any licence fee! Anyway, there are countries where you cannot protect your patents. Maybe there will be better to make technology open from the beginning?

Aubrey de Grey: I think you’ve pretty much answered your own question with that quote. The technologies that will drive rejuvenation are not so easy to suppress.

Ariel Feinerman: Are SENS RF going to begin new research programmes in 2019?

Aubrey de Grey: Sure! But we are still deciding which ones. We expect that our conference in Berlin (Undoing Aging, March 28-30) will bring some new opportunities to our attention.

Ariel Feinerman: What are your plans for 2019?

Aubrey de Grey: I’d like to say less travelling, but that doesn’t seem very likely at this point. Really my goal is just to keep on keeping on – to do all I can to maintain the growth of the field and the emerging industry.

Ariel Feinerman: Thank you very much for your answers, hope to see you again!


Watch the video: Γιατί μάλλον Δεν Είμαστε ο Εξυπνότερος Λαός - ft. @Καθημερινή Φυσική. Greekonomics #21 (July 2022).


Comments:

  1. Faine

    Very useful thought

  2. Gardagor

    the response)))

  3. Yoktilar

    It is necessary to be optimistic.

  4. Garwood

    Even so

  5. Fausto

    I apologize, but it's not quite what I need.

  6. Kajikora

    Instead of criticising advise the problem decision.



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