VDP-61 [Funding]: Boston Matrix

[rpill]

One-liner: Discovery and development of a novel antiglycation and anti-crosslinking agent aimed at decreasing ECM stiffness.

This proposal is based on the supporting documents provided by Dr. Roman Litvinov and Alexey Strygin, as well as questions and answers to their team, and senior reviews.

Longevity dealflow WG team

Initial evaluation: Joppe Nieuwenhuis, Diane Seimetz, Anonymous reviewer (prof.)
Final evaluation: Diane Seimetz, Tim Peterson, Nina Patrick, Jean Hebert

Sourced by/Shepherd: Rakhan Aimbetov
Squad members: Estefano Pinilla, Paolo Binetti, Max Unfried, Laurence Ion
Project PI: Dr. Roman Litvinov

4/4 senior reviewers have expressed a vote in agreement to staged funding for this proposal. Here is the digest:

Quantitative reviews:

To quantify the level of conviction, the reviewers provided a score on a scale of 1-5 (with 5 being the highest).

The average score was 2.9/5.0.

Brief qualitative review summaries:

Under consideration of the various discussions and potential concerns, the proposed approach to de-risk the project by a staged approach starting with a $30k fund to conduct a short PoC study in an experimental glycation animal model is endorsed. For the decision making on the follow-on funding, the totality of data, i.e. from the animal study as well as the in vitro data, should be considered.

I favor funding the first two milestones up to $155K but I recommend any additional funds should require external capital.

The staged financing makes sense, which I endorse. It’s more likely with the current in vivo testing focus that whatever they end up testing has a positive health effect, regardless of glycation status or mechanism of action, and so even if it means a pivot, may have commercial value.

I am supportive of the first financing tranche of €30K for the team to demonstrate traction and de-risk the project to attract outside investment.

Simple summary

Intra- and intercellular components constantly get non-enzymatically modified by simple sugars and reactive glycolytic metabolites in a process known as glycation. This is especially true for long-turnover proteins such as collagen, elastin, fibronectin, etc. found in the extracellular matrix (ECM). Advanced glycation end products (AGEs) that form as a result of glycation often exist as irreversible crosslinks between ECM components and/or as adducts on ECM molecules. The accumulation of AGEs leads to the loss of matrix elasticity and its stiffness, as well as the activation of receptors for damage-associated molecular patterns (DAMPs), mechanistically linking glycation to a wide range of deregulatory processes characteristic of old age and associated diseases. Some reports propose ECM glycation and downstream effects (stiffness, etc.) as a new hallmark of aging that drives cellular senescence, stem cell exhaustion, chronic systemic inflammation, and other pathogenic processes.

Notably, elevations in AGEs with consequent collagen-based cardiomyocyte and extracellular matrix stiffness [link], as well as glycation-related aseptic inflammation [link], are among the pathogenetic mechanisms of diabetic cardiomyopathy (DbCM). The project is aimed at screening a library of natural plant extracts for the discovery of novel compounds that would inhibit the ECM glycation process within the context of DbCM as a surrogate indication.

Requested funding

As discussed at longevity dealflow working group meetings and in the designated thread in the WG Discord channel, in order to critically evaluate the project’s feasibility and attractiveness in terms of fundability, the applicant team is asked to show proof-of-concept (PoC) efficacy of extracts in an in vivo model. Consequently, the team is asking $30k (see Experimental plan and Financing and milestones/First financing stage: $30k sections below) to conduct a short PoC study in an experimental glycation animal model.

If the PoC experiments show sufficient efficacy of extracts, the proposal will be re-voted on with the full experimental and IP set-up in scope.

VitaDAO’s available funds

For context, VitaDAO funded 15+ projects with $3.5m+, and has ~$4.5m in liquid funds remaining (before further fundraising), which will be used for:

1. Funding new projects (such as this)
2. Operations, including sourcing, incubation, evaluation, & community growth
3. Follow-on funding, including for projects VitaDAO will spin out

Problem

Natural aldehydes and ketones such as glucose, fructose, and galactose, as well as reactive dicarbonyls glyoxal and methylglyoxal, react with biological amines – e.g. N-terminal and side-chain amino groups in amino acids, phosphatidylethanolamine, phosphatidylserine – to form various adducts and crosslinks. These non-enzymatic modifications, given enough time, become irreversible and constitute so-called advanced glycation end-products (AGEs).

AGEs accumulate with age and contribute to age-related pathologies via several routes:

• Adducts such as carboxyethyllysine (CEL), carboxymethyllysine (CML), fructosyl-lysine, etc, bind to receptors of the RAGE (receptors for AGEs) family and stimulate inflammatory reactions.

• Modified proteins, on the other hand, sustain disruption of their tertiary structure and elicit unfolded protein response which, if unchecked, can contribute to the loss of proteostasis observed during aging and diseases like Alzheimer’s, Parkinson’s, etc.

• Crosslinks such as glucosepane, pentosidine, MOLD, MODIC, etc. covalently tether proteins together interrupting their normal function.

For intracellular proteins, the aforementioned modifications are bearable since most proteins have a relatively fast turnover rate and their pool is constantly replenished (although there is evidence for histone glycation and subsequent negative consequences). However, in the case of long-lived proteins, glycation can have a prolonged detrimental effect. This is especially true for proteins of the ECM because these proteins have half-lives which are considerably longer.

Crosslinking of ECM proteins – collagen, fibronectin, elastin – leads to the rigidity of the matrix. The accumulation of crosslinks has a profound effect on blood vessel elasticity and places an increasing strain on the cardiovascular system. Moreover, several reports show that stiffness of the ECM is the sole factor that can promote cellular senescence and dampen stem cell differentiation.

There are several means to ameliorate the effects of ECM glycation. Firstly, it is to break existing crosslinks either enzymatically – the approach currently developed by Revel Pharmaceuticals. However, given the multitude of various crosslinks and adducts, this approach requires an array of interventions each aimed at a specific glycation end product.

An alternative approach is to attenuate glycation, and this modality has been tried with the compound aminoguanidine. This synthetic compound, however, did not pass clinical trials due to safety concerns. Since naturally-derived therapeutic molecules generally have better safety profiles, it is reasonable to turn to natural sources for the discovery of new drugs with antiglycative characteristics.

It should also be noted that in a recent paper – “New hallmarks of aging” – glycation-associated ECM stiffness is considered both a hallmark and one of the pathogenetic mechanisms of aging. This adds to the importance of having interventions targeting glycation in the portfolio of longevity assets.

There are 12.9M patients with DbCM in the 7 largest Western Nation countries. Current treatment approaches focus on clinical manifestations of DbCM and hemodynamic disturbances rather than on pathogenetic mechanisms. One of the approaches with good efficacy evidence [link 1, link 2] is SGLT-2 inhibitors. They have decent efficacy in the prevention of CVD-related deaths and hospitalizations, a decent safety profile, and medium-to-high costs. If at least one of these parameters can be improved upon, there is a good chance to capture a considerable share of this market.

Hypoglycemic agents with cardioprotective properties, such as SGLT-2 inhibitors, etc., have limited potential as longevity therapies due to the high likelihood of causing hypoglycemia in non-diabetic patients. They argue that if our candidates exhibit a substantially improved safety profile, even bigger market (prevention of diabetic cardiomyopathy in high-risk patients diagnosed with type 2 diabetes (T2D), metabolic syndrome, or prediabetes) will become available.

Opportunity

Plant-derived polyphenols (extracts) were shown to be effective against various cardiomyopathies including DbCM, combining different pro-longevity mechanisms of action (MoA), such as mTOR regulation and modulation of its downstream effectors (not only antiglycation activity).

The Boston Matrix team is planning to screen over 2500 natural plant extracts, isolate and develop a novel therapeutic candidate. The MoA would include antiglycation activity, prevention of ECM cross-linking, and amelioration of ECM stiffness. Previously, they have used this approach to sсreen for a number of existing natural compounds/extracts and novel chemically synthesized molecules.

The team has access to a collection of 2500+ natural extracts (and means to identify, isolate and modify active ingredients to make patentable products with drug-like properties) and to a library of 500+ natural compounds (mostly novel) already isolated from these extracts. Additionally, they have an in silico platform and tools for the discovery and optimization of ECM stiffness-ameliorating molecules. The platform and the tools are trained on 199 molecules (isolated, modified, or chemically synthesized) with a proven antiglycation effect [Russian patents RU2021623030, RU2021622654].

Most natural plant extracts contain polyphenols, flavonoids, anthocyanidins, and other active compounds. Preliminary trials have shown that quercetin, luteolin, and tannin possess remarkable antiglycation activity in in vitro tests done in their lab (IC50 for quercetin is ~3 μg/ml; for tannin – less than 1 μg/ml). Green tea and grape seed extracts have high concentrations of polyphenolic compounds and also show high antiglycation activity (3-6 μg/ml). The initial screens of the first 50 of 2500 extracts had shown leads with antiglycative properties far exceeding those of quercetin and the reference compound aminoguanidine [report0]. Subsequent studies identified several leads that exceed the stated IC50 value (Chamarhodos sabulosa, Swida sanguinea, etc. [report1]). Additional research has shown that lead extracts were able to stimulate mitochondrial metabolic processes (as judged from the MTT assay) exerting cytovitalic (opposite to cytotoxic) action [link]. At the same time, reference antiglycator quercetin elicits cytotoxicity in the same concentration range.

This and the whole history of pharmacology suggests that plants are a great source of efficacious and safe compounds. The applicant’s philosophy for preclinical development is based on testing the activity of compounds in ever more complex (and costly) test systems weeding out unpromising candidates along the way. They will use the simplest in vitro tests modeling pathology (in this case – the glycation process) for initial screening. After that, the leading candidates will be tested in cellulo. They do not plan in vivo studies validating the ability of lead isolated compounds to suppress cardiomyopathy in diabetes or prolong life in laboratory animals at this stage as they will not be able to fit the trials in a 12-month timeframe and in the budget. However, to determine the ability of lead extracts to act as a source of potential antiglycating agents, it is planned to utilize a quick in vivo model that verifies the ability of the extract components to penetrate the biological barrier of the gastrointestinal tract and remain stable in amounts sufficient to exhibit antiglycation activity. The model assumes subchronic methylglyoxal intoxication (intraperitoneally, 17.25 mg/kg, equivalent to that produced in diabetics) combined with oral administration of an extract.

Potential non-aging indications for regulatory approval are T2D and its long-term complications (nephropathy, angiopathy, etc., as well as cardiomyopathy, selected as a surrogate indication), cardiovascular diseases, Alzheimer’s disease, and other proteopathic neurodegenerations (in complex treatment). Targeted hallmark – loss of extracellular proteostasis (ECM cross-linking is considered a stand-alone hallmark of aging; link). Other possible applications of the platform with faster time-to-market are the cosmetic dermatology industry, cosmeceuticals, and nutraceuticals.

The described preliminary work was obtained by scientific groups which included Roman Litvinov, and was published in peer-reviewed journals [link1, link2, link3].

Evidence supporting the concept that increased ECM stiffness is associated with glycation [link 1, link 2], can negatively affect cell and tissue properties [link], and has a negative impact on longevity [link] is plentiful. The most relevant manifesto reflecting this notion has been published in the recent “New hallmarks of aging”:

“Altered mechanical properties apply both to cells and to the extracellular milieu … Finally, the extracellular matrix also changes with ageing, which greatly alters cell behavior. Increased rigidity and loss of elasticity, for example arising through glycation cross-links between collagen molecules, can lead to multiple age-related disease states such as hypertension with concomitant kidney and neurological defects – such cross-linking may contribute to the accelerated aging seen in patients with diabetes”.

The above quote also confirms the appropriateness of choosing CVDs (and DbCM in particular) as a target indication since matrix crosslinking contributes to increased vascular stiffness, abnormal vascular response, arterial hypertension, and (eventually) cardiac pathology. Outside the context of aging, this mechanism is particularly relevant to diabetes.

Target product profile

Indication	Diabetic cardiomyopathies
Target population	- Patients with T2D and diabetic cardiomyopathy - Patients with T2D and at risk of CVDs (label extension) - Patients at risk of prediabetes, metabolic syndrome, atherosclerosis and exposure to high-AGE food (longevity label extension)
Therapeutic modality	Small molecule (isolated from natural extract or modified)
Efficacy	Primary outcomes: - 40%+ lower 3-year risk of CVD-related death vs placebo - 35%+ lower 3-year risk of heart failure hospitalisation Secondary and label extension outcomes (% changes TBD): - Lower progression stage B to overt heart failure (stage C) - Percentage of blood ejection before and after 12 weeks of treatment - Change of LVEF between before and after 12 weeks of treatment - Peak VO2 during cardio-pulmonary exercise test (CPET); [15 months after randomization] - Change in myocardial perfusion reserve index calculated from cardiac MRI - HbA1c, general fluorescent AGEs level (which includes pentosidine, vesperlysines A, B, C, crossline, fluorolink, FFI etc.) - Pulse-wave velocity - Ankle-brachial index
Safety	Generally very well tolerated (important for longevity). Incidence of adverse events: - Hypoglycemia (<1%) - UTIs (<5%) - Nausea (<1%) - Upper respiratory tract infection (<2%)
Administration	Per os
Mechanism of action	Anti-glycation agent
Biological activity	- Lower hypertension incidence due to less of vascular fibrosis and AGEs in the vasculature (i.e. less stiffness of vessels) - Lower tissue levels of AGEs Thus, systemic effect on cardiomyopathy progression.

Experimental plan

Stage 1 (already started and ongoing):

• Preparation and initial screening of 2500+ natural plant extracts to assess their antiglycation activity (~4-5 months):

The purpose of this stage is to assess the antiglycation activity of 2500+ natural extracts rapidly and cost-effectively in an assay modeling accelerated glycation. The team will monitor glycation end products (for all extracts) and intermediate products of glycation (for lead extracts). Progressively, as they weed out more extracts, they will make the assay more complex by introducing additional parameters (e.g. temperature – increased to accelerate glycation/equal to body temperature; ion composition – presence/absence of transition metals). This is aimed at improving the specificity of the results (variations in the buffer used, temperature, glycating agent, glycated protein, etc., as well as the detection method, are required in order to more fully describe dominant mechanisms of antiglycation activity and confirm the activity of leads). Cytotoxicity studies of the most promising candidates are also planned for this stage.

Stage 2:

• Short-listing and further analysis of lead natural extracts. Confirmation of the antiglycation activity of the leading extract in an in vivo model. Initial talks with patent attorneys. [additional 2-4 months after the end of stage 1]:

1. additional assessment of antiglycation activity using ELISA
2. exploration of antiglycation activity mechanisms (antiradical, carbonyl scavenging, metal chelation)
3. short in vivo model to confirm the activity in living organisms

ELISA with anti-AGEs antibodies will be used to confirm the results of in vitro. The dominant mechanism of the leads’ antiglycation activity is planned to be studied using a triplet of tests (antiradical activity, carbonyl scavenging, inactivation of transition metals). The activity of the best leaders will be tested in a brief animal model to evaluate the ability to suppress the advanced glycation induced by methylglyoxal subchronic intoxication. This test will be used to confirm the activity of extracts in an animal model (PoC).

Stage 3:

• Testing the activity of isolated active compounds (3-4 months after stage 2):

4. preparative chromatography
5. additional exploration of antiglycation activity and detailed mechanisms thereof
6. exploration of complementary activities (carbonyl scavenging, transition metals scavenging, antiradical activity, etc.)
7. cytotoxicity evaluation
8. analysis of markers associated with cellular response to glycation products (MMPs, ILs)

• Optimizing the lead candidate structure via the in-house developed AI-powered tool and medicinal chemistry techniques. Optimized structures will not be tested for activity at this stage. However, it still will be possible to protect the most promising compound and derivatives.

• Preparation of materials for patenting (single or combined identified compound(s) and(or) virtually assessed compound and (if relevant) derivatives thereof) and subsequent scientific publication.

As the most promising extracts are selected, the team will be identifying active ingredients in these extracts by LC-MS. When the contents of extracts are identified, the team plans to apply preparative chromatography techniques and test the activity of isolated compounds repeating stages 1-2.

Promising leads will be tested in cellular models to determine the ability to influence the important markers of aging and DbCM pathogenesis. The team plans to test a relatively narrow range of markers that change due to glycation. The long-listed markers are cellular AGE levels, inflammatory cytokine levels (IL1b, IL6, IL8, TGFb, and other), metalloproteinase levels (MMP2, MMP9, TIMP1, and other). The final choice of markers will depend on the results of the first experiments. Part of these trials is planned to be outsourced. Considering the groupability of markers, if one of the two groups of markers is successfully influenced, the analysis will be expanded towards this group of markers. At present, it is impossible to predict with sufficient accuracy which particular group of markers will be affected by a particular compound.

If necessary, in silico optimization of most promising leads (based on quantum chemical QSAR-analysis neural network optimized for improving antiglycation effect – trained on datasets already available and those that will be generated during the experiment) will be applied.

Description of the assays and methods

In their work, the applicants use screening of a wide range of candidates, highlighting the main and additional methods of research.

The whole-extract antiglycation activity screening assay was devised by the team and published in peer-reviewed journals [link 1, link 2, link 3].

The initial screening technique is based on the reaction of glycation of albumin (the main model protein) with glucose (a common glycation agent) with applied heat (to stimulate the reaction) and detection of fluorescent glycation products at 5 or 10 different pairs of excitation/emission wavelengths; 2 pairs of excitation/emission wavelengths corresponding to fluorescent amino acids and some additional parameters (if necessary). The approach was preliminarily characterized here.

A large number of wavelength pairs correspond to different AGEs (namely pentosidine, vesperlysine A and B together, vesperlysine C, fluorolink, FFI, crossline, lysyl-pyrrolidine), and some oxidation products are used for minimization of interference from the test extractives. The activity results estimated at some wavelengths are not taken into account if the extracts show the ability to quench fluorescence at this pair of wavelengths. Only data at non-interfering pairs of wavelengths are used. Additional assessment of the amino fluorescence (namely, tryptophan and tyrosine) is aimed at testing the ability of the compound to prevent the loss of the native protein conformation during the glycation reaction. This provides a reliable result of the initial screening of the antiglycation activity.

The estimated bandwidth of the initial antiglycation quantification model is at about 2500 extracts/5 months. To increase the assay throughput, the glycation reaction will be sped up by heating the reaction mixture to 60C in PBS pH 7.4. The team had determined all key parameters of their model, reflected in the Methods section of their first reports [report0, report1], and in one of the demo study protocols [link].

The condition for passing the screening will be an IC50 of less than 3 μg/ml (at present, the applicants have already established several extracts with IC50 values as low as 0.4 μg/ml, which means that the first milestone is achieved: Chamarhodos sabulosa IC50 0.4-1.4 ug/ml, Swida sanguinea IC50 ~0.46 ug/ml, Sedum hybridum IC50 ~0.37 ug/ml, Oenotera biennia IC50 ~0.7 ug/ml [report1] (IC50 values will be updated because some values in these initial studies were extrapolated). The 3 ug/ml concentration in the first milestone had been included based on the activity of green tea extract with high (95%) content of polyphenolic fraction which contains a strong antiglycation agent epigallocatechin gallate. As a reference, they use functionally, and structurally close compounds (tannin, quercetin), as well as the well-known antiglycation/deglycation molecules some of which were evaluated in clinical trials as anti-AGE agents (aminoguanidine, alagebrium, etc.), and molecules used clinically for other indications with known antiglycation activity (pyridoxamine, hydralazine, etc.).

Further analysis allows the possibility of testing the activity using proteins other than albumin (gelatinized collagen) and glycation inducers other than glucose (methylglyoxal, glyoxal, fructose, etc.) – as an example, a protocol for studying albumin glycation with various glycation agents at two different temperatures is attached [link] – as well as probing the activity of the lead extracts in the test system using ELISA and glycation temperatures equal to the human body temperature. At body temperature, the ELISA assay will require extended incubation (for 28 days) of reaction mixes since the ability of antibodies to detect AGEs diminishes if glycated albumin had been heated to 60C. The results of tannin’s antiglycation activity assessed in the 28-day model (fluorescence in 5 concentrations, ELISA in 2 concentrations) is available via the following link [link].

Parallel to finding the most promising extracts, they also plan to perform preliminary analyses of the extracts’ dominant MoA zooming in on three modalities: the ability to scavenge carbonyl compounds, the ability to bind transition metals, and their antioxidant action. All mentioned models are fine-tuned, methodologies described [link 1, link 2, link 3].

Moreover, they also plan to determine the polyphenolic content of our extracts, since all well-known plant bioactive compounds with antiglycative properties belong to the class of polyphenols [link].

The study is expected to be combined with an assessment of toxicological properties in cell cultures. A sample protocol describing cytotoxicity assay in HepG2 cells using MTT is accessible via [link]. According to preliminary data, some lead extracts – possessing antiglycation activity in the ng/ml concentration range – possibly are able to stimulate mitochondrial metabolic processes (exerting cytovitalic, i.e. not cytotoxic, action). NB: the medium containing the extract was removed before adding the MTT reagent, therefore the observed effect is most likely not due to extract interference. At the same time, the reference compound quercetin elicits cytotoxicity in the same concentration range.

The lead activity will be evaluated in an in vivo model designed to test for the capacity to suppress the advanced glycation, induced by subchronic methylglyoxal intoxication. They have devised a technique that allows for complex analysis of compounds’ antiglycative properties. The technique entails subchronic intoxication with methylglyoxal (17.25 mg/kg intraperitoneally, which is equivalent to amount generated in diabetic patients) combined with oral administration of tested extract/compound [link 1, link 2]. Since molecules absorbed from the visceral peritoneum, mesentery, and omentum are drained into the portal vein, whereas molecules absorbed from the parietal peritoneal capillaries and lymphatic vessels are drained directly into systemic circulation [link], the experiments will be concluded by extracting liver autoptates and serum collection followed by a comparative quantification of the AGE content. The model has been calibrated by the scientific team using ALT-711 as a reference compound and can be applied to testing the whole extracts’ bioavailability, activity retention in the biological milieu, and suppress glycation in vivo (PoC).

For compound isolation, they will utilize preparative chromatography coupled with validation by mass spectrometry. First of all, they plan to check the activity of all (or at least the most promising) isolated compounds in a battery of screening assays as described for whole extracts. It’s entirely possible that at this stage they’ll show that varying activity components are associated with distinct compounds. Should this be the case, they’re considering the possibility of compound combinations.

In the assays employing cell cultures, in addition to the direct detection of AGEs, an immediate marker of glycation, they plan to carry out the assessment of the influence of substances under study on the production of MMP2 and MMP9, as well as IL-1b and IL-6, by fibroblasts in response to the glycated cellular environment. These molecules serve as important predictors of myocardial remodeling and associated negative outcomes [link 1, link 2]. The said markers are either immediate ECM remodelers (metalloproteinases) or markers of aseptic inflammation indirectly linked to remodeling; if any of the groups shows to be regulated by glycation, they’ll expand their analyses in the direction of the affected group. Presently, it’s impossible to predict which markers will be affected by any given substance.

They have defined a surrogate indication – diabetic cardiomyopathy. The proposed markers are prominent in both the aging process and the surrogate indication. The markers can be divided into two groups: 1. linked to aseptic inflammation and inflammaging (pathogenically associated with aging [link] as well as with diabetic cardiomyopathy [link], and elicited by AGEs), such as IL-1, IL-6, IL-8, TGF-b, etc.; 2. linked to matrix remodeling (associated with aging [link] and diabetic cardiomyopathy [link], and brought about by AGEs in the ECM), such as MMP2, MMP9, TIMP1, etc.

At the final stages of the project, they will take advantage of our in silico platform – in order to ensure the patentability of the result (not for screening purposes). A compound modeled using the quantum chemical QSAR model will have a greater potential to become an IP than an isolated natural substance. All the while, in silico tests are optional because increasing patentability prospects may not be required if an isolated active substance itself – as decided by a patent attorney – will be the object of IP.

Limitations: whole extracts or isolated components of extracts can be difficult to patent. This limitation is addressed by introducing the QSAR neural network-based lead optimization, as well as collaborations with synthetic chemistry experts to model the potential modification (virtual leads) and assess the technical and economic viability of synthesis/production.

IP roadmap

Since Open Longevity (OL) – a parent company for Boston Matrix – is non-profit, the applicant team is not positioned to develop a commercial product beyond discovery and PoC trials. The team plans to start negotiating patenting options upon receiving their first results as indicated in the work plan (stage 2) and file for a patent concluding the experimental part of the project (stage 3). The team will require assistance from VitaDAO in filing for a patent and sees the DAO as the IP holder. Further IP strategy might include protection of an optimized lead, use of lead(s) vs particular indication(s), formulation and production process.

This application is the first stage of the ROADMAP to solving the problems of ECM stiffness and glycation in aging. The team expects more opportunities for IP generation to become available in the future.

Team

Roman Litvinov, M.D., Ph.D. in pharmacology and clinical pharmacology, head of group. Academic positions: senior researcher, assistant professor. Scientific interests: molecular biology of glycation reaction, pharmacological control of glycation reaction, advanced glycation end product (AGE) inhibitors, crosslink breakers, receptor for AGEs (RAGE), AGEs-associated pathologies, metabolic disorders. Several molecules Roman has worked on proceeded to clinical trials.

Alexey Shavarda, Ph.D., provides a unique collection of 2500+ plants from territories of Eurasia: Kazakhstan, Kyrgyzstan, and Russia.

Alexey Strygin will be responsible for turning the project into a commercializable IP, business networking, and organizational support.

Umida Ibragimova, researcher, PhD student.

Alina Rzaeva, researcher, PhD student.

Nikita Valuysky, researcher, student.

Marina Gasheva, researcher, student.

Angeline Shushakova, researcher, student.

Open Longevity is a non-profit that possesses a vast network of longevity scientists, engineers and enthusiasts, and encompasses a number of initiatives aimed at accelerating aging research. OL team members will be responsible for general project management and coordination, PR, and IR: Mike Batin (founder), Anastasia Egorova (CEO), Timofei Glinin, Ph.D. (CSO)

Budget

Item	Cost
Extract preparation	$25-40k
Initial screening in in vitro assays of glycation, insights into mechanisms, initial in vivo study	$40-55k
Secondary screening of most promising candidates (initial mechanism of action of the leader extracts)	$40-50k
Isolation, identification of active ingredients of short-listed candidates	$15-25k
Initial mechanism of action and cytotoxicity studies (in partner lab or CRO)	$100k
Patenting and other various costs	$10-15k
Video filming and production	$15k
Total	$300k

Financing and milestones

First financing stage: $30k

• PoC in an in vivo model of extracts’ antiglycation activity.

Second financing stage: $115k

The funding is contingent on the lead extracts’ performance in vivo on par with the reference compound quercetin.

Milestone:

• A short list of candidate extracts (1 or more) with antiglycation activity (IC50 3 μg/ml or less; if translated to humans it would roughly approximate to the effective dose of 150-300 mg of unmetabolized compound taken orally assuming uniform distribution), initial assessment of MoA and verification of antiglycation activity in the PoC in vivo model.

Third financing stage: $155k

Milestones:

• 1 or more isolated compounds with high antiglycation activity. Detailed MoA and pharmacological properties;

• Filed patent application to protect the above-mentioned compound(s);

• A video documentary (no less than 20 minutes long) describing all the development stages is filmed and edited. VitaDAO funding is mentioned in the beginning and in the end of the documentary.

Important note: due to the separation of the project into 3 milestone groups, the overall timeline might increase as some processes that could have been done in parallel will be performed sequentially.

Strengths

• The group possesses unique experience in a relatively new field – glycation reaction and crosslinks as a target for longevity interventions.

• Their preliminary PoC data show a promising antiglycation effect of several natural plant-derived compounds.

• In vivo experiments to confirm the antiglycation activity of extracts will be conducted.

• IC50 of these compounds is two orders of magnitude lower than the reference antiglycation compound aminoguanidine and up to 10 times more active than quercetin which suggests a better safety profile.

• The team has access to the Open Longevity talent pool.

Risks

• The initial screening will be done in vitro. Lead molecules might not be as effective in vivo (the risk will be addressed by the addition of PoC tests in an in vivo model).

• The inhibition of glycation could have no effect on already glycated macromolecules, therefore only delaying damage accumulation rather than repairing (despite the possibility of limited efficacy, the team has a general roadmap that has the potential to be effective against already formed advanced glycation end products, and the current project is the first stage of that strategy).

• It might not be possible to isolate active compounds or isolated compounds will not be amenable to synthesis (the risk will be mitigated due to the expectedly large number of leading extracts).

• There’s a risk of not procuring sufficient contingent funding for the Third financing stage.

• The war between Russia and Ukraine may interfere with the applicant team’s ability to make progress (harder access to consumables/need to relocate the lab).

At present, the team asks for $30k to show PoC efficacy of their extracts in an in vivo murine model. Please vote below if you think the team should be funded with the requested amount.

• Agree
• Agree with revisions (please comment)
• Disagree

[rpill]

The proposal will be repositioned towards diabetic cardiomyopathy as a surrogate indication. However, please feel free to leave your comments so that the applicant is able to see them and respond.

[bowtiedshrike]

This looks like a high risk fishing expedition with a poor plan of execution.

The screening assay is not described at all. Not clear how high throughput this assay is, or how well it works. Without preliminary experimental (not in silico!) data showing the screening assay works, this project is NGMI. If they can’t get the screen to work, there is no project. If they’re not clear what temp, pH, buffer they want, it does not bode well for this assay. Those should already be optimized. Same with the ELISA approach if they plan to use that to confirm their hits. Show positive, negative controls and a titration of one plant extract that has a known AGE blocker like quercetin or tannin.

Stage 2 is also poorly defined. The markers should be defined no matter what plant extract comes up in their screen. None of the proposed markers will address mechanism. Cytokines are not long-lived components of the ECM, so unclear why those are tested. Also, how did RAGE fail to make that list?

The active might be a mix of compounds. No plans to consider that.

The IC50 for the first milestone is rather high–the IC50 milestone has already been achieved by existing molecules.

It is unclear what quality this ‘plant extract library’ is. Have these extracts been validated for cytotoxicity? Are they water soluble? Do they have a realistic chance of being stable/delivered even if they work in the functional assays?

It’s not clear from the budget how many people of what qualifications are working on the project, or if it’s all out-sourced, or supply costs. If they want $25-$40k to prepare the extracts, they don’t have the extract library in hand. If they have problems at that stage, whole project is sunk.

Why is $15k going to a video documentary?

I thought patenting in the US and Europe ran closer to $30k than $10-$15k. Also, small molecules also have shorter patent times in the US now due to the Inflation Reduction Act. This will reduce the value of any IP generated around these molecules even if successful.

[longevion]

Added a poll. The project is still open for revision suggestions / incubation.

[rpill]

Hi, @longevion! The text is going to change to include the surrogate indication. Waiting for Boston Matrix to approve the final version before posting.

[MAC]

@rpill

An alternative approach is to attenuate glycation…

What about donating blood, phlebotomy to dump old glycated junk? I have been donating blood (450mL) every 8 weeks for almost 5 years. Turning over complete whole body blood every 18 months approx. There are a plethora of vascular and possibly immuno benefits as well to phlebotomy.

[rpill]

Hello, @bowtiedshrike! Please see below the response to your comment from Boston Matrix:

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Dear commentator, we’re thankful for the opportunity to provide a comprehensive response to your comment. After all, critical evaluation of projects is the strongest feature of open science.

@bowtiedshrike

The screening assay is not described at all.

We’ve based our screen on the whole-extract antiglycation activity assay devised by us and published in peer-reviewed journals [1,2,3].

@bowtiedshrike

Not clear how high throughput this assay is, or how well it works.

We estimate the bandwidth of the initial antiglycation quantification model at about 2500 extracts/5 months – the estimate established on the basis of extensive preliminary work (the experimental work experience of GLP-certified applicants in regard to the basic screening assay is 8 years.) To increase the assay throughput, we speed up the glycation reaction by heating the reaction mixture to 60C in PBS pH 7.4.

@bowtiedshrike

If they’re not clear what temp, pH, buffer they want, it does not bode well for this assay.

We had determined all key parameters of our model, reflected in the Methods section of our first report [link] and in one of the demo study protocols [link]. The model is validated, its precision varies in the range of 4-12% (RSD%).

Additionally, the report contains antiglycation data on 69 extracts from our collection. The quantities measured so far indicate that we’ve already found a few samples with antiglycative properties exceeding those of the established reference compounds such as quercetin, and achieved the first milestone set forth in our proposal – i.e. finding lead extracts with IC50 less than 3 ug/ml.

Parallel to finding the most promising extracts, we also plan to perform preliminary analyses of the extracts’ dominant mechanism of action zooming in on three modalities: the ability to scavenge carbonyl compounds, the ability to bind transition metals, and their antioxidant action. All mentioned models are fine-tuned, methodologies described [1,2,3].

Moreover, we also plan to determine the polyphenolic content of our extracts, since all well-known plant bioactive compounds with antiglycative properties belong to the class of polyphenols [link].

@bowtiedshrike

Same with the ELISA approach if they plan to use that to confirm their hits. Show positive, negative controls and a titration of one plant extract that has a known AGE blocker like quercetin or tannin.

As we determine lead extracts in our collection, their activity will be characterized further in a prolonged glycation model, which requires extended incubation (for 28 days) of reaction mixes at body temperature (the ability of antibodies to detect AGEs diminishes if glycated albumin had been heated to 60C). The results of tannin’s antiglycation activity assessed in the 28-day model (fluorescence in 5 concentrations, ELISA in 2 concentrations) are available via the following link [link]. The 28-day incubation tests have been carried out only preliminarily – solely using the reference compound – in order to confirm our working concept; the characterization of extracts – including titration of active concentrations – will be done in the future. Any differences in activity estimates in different assays are due to distinct detection techniques and the AGEs detected.

@bowtiedshrike

Without preliminary experimental (not in silico!) data showing the screening assay works, this project is NGMI.

Separately, we would like to note that our in silico platform will be utilized only at the finalizing stages of the project – in order to ensure the patentability of the result. A compound modeled using the quantum chemical QSAR model will have a greater potential to become an IP than an isolated natural substance. All the while, in silico tests are optional because increasing patentability prospects may not be required if an isolated active substance itself – as decided by a patent attorney – will be the object of IP.

@bowtiedshrike

This looks like a high risk fishing expedition with a poor plan of execution.

Although you defined our project as a high-risk fishing expedition, according to our earlier report we have already identified three extracts showing high activities which may not be limited to solely antiglycation. Some lead extracts possess antiglycation activity in the ng/ml concentration range (Chamarhodos sabulosa IC50 0.4-1.4 ug/ml, Swida sanguinea IC50 ~0.46 ug/ml, Sedum hybridum IC50 ~0.37 ug/ml, Oenotera biennia IC50 ~0.7 ul/ml [link]) and, possibly, are able to stimulate mitochondrial metabolic processes exerting cytovitalic (not cytotoxic) action. The latter is presented in the following protocol [link]. At the same time, reference compound quercetin elicits cytotoxicity in the same concentration range.

@bowtiedshrike

Stage 2 is also poorly defined. The markers should be defined no matter what plant extract comes up in their screen.

We’ve outlined our stage 2 working plan in the proposal. We plan to carry out the analysis starting with assessing the influence of substances under study on the production of MMP2 and MMP9, as well as IL-1b and IL-6, by fibroblasts in response to the glycated cellular environment. These molecules serve as important predictors of myocardial remodeling and associated negative outcomes [1,2]. The said markers belong to two groups – immediate ECM remodelers (metalloproteinases) and markers of aseptic inflammation indirectly linked to remodeling; if either of the groups shows to be regulated by glycation, we’ll expand our analyses in the direction of the affected group. Presently, it’s impossible to predict which markers will be affected by any given substance.

@bowtiedshrike

None of the proposed markers will address mechanism. Cytokines are not long-lived components of the ECM, so unclear why those are tested.

We disagree that none of the proposed markers address the mechanism. As a result of our discussions with the VitaDAO community, we’ve defined a surrogate indication – diabetic cardiomyopathy. The proposed markers are prominent in both the aging process and the surrogate indication. The markers can be divided into two groups: 1. linked to aseptic inflammation and inflammaging (pathogenically associated with aging [1] as well as with diabetic cardiomyopathy [2], and elicited by AGEs), such as IL-1, IL-6, IL-8, TGF-b, etc.; 2. linked to matrix remodeling (associated with aging [3] and diabetic cardiomyopathy [4], and brought about by AGEs in the ECM), such as MMP2, MMP9, TIMP1, etc. Additionally, we will directly quantify the level of AGEs inside cells, which is a proximate criterion of advanced glycation.

@bowtiedshrike

Also, how did RAGE fail to make that list?

We acknowledge the importance of RAGE overexpression in response to glycation- and AGE-derived DAMPs. However, in contrast to cellular markers of inflammation (interleukins) and matrix remodeling (metalloproteinases) – who act directly either on cells or the ECM respectively and usually are not used as targets – RAGE is not only a marker for advanced glycation but also itself is a therapeutic target. Downregulation of RAGE is possible not only as a result of glycation inhibition but the receptor blockade and downstream signaling attenuation too. With the current project, we did not include RAGE as a supplementary target because that would lead to an additional budget increase.

@bowtiedshrike

The active might be a mix of compounds. No plans to consider that.

At the active substance isolation stage, we plan to check the activity of all (or at least most promising) isolated compounds in a battery of screening assays as described for whole extracts. It’s entirely possible that at this stage we’ll show that varying activity components are associated with distinct compounds. Should this be the case, we’re considering the possibility of combining compounds. We should note that not only antiglycation might be complemented by other protective mechanisms associated with various compounds present in extracts, glycation inhibition itself could be realized by a range of mechanisms, such as protein amino group protection, transglycation, antioxidant action, amadorin activity, dicarbonyl scavenging, chelation of transition metal cations, etc. If we establish that different observed activities are due to distinct isolated compounds, the latter will be combined.

@bowtiedshrike

The IC50 for the first milestone is rather high–the IC50 milestone has already been achieved by existing molecules.

The 3 ug/ml concentration in the first milestone had been included based on preliminary data summarized in the Boston Matrix’s first report [link] and suggestions from VitaDAO. This IC50 was demonstrated for a green tea extract with high (95%) content of polyphenolic fraction which contains a strong antiglycation agent epigallocatechin gallate. Notably, IC50 depends not only on components’ activity but on their concentrations as well. Therefore, the proposed value allows considering an extract as a lead in case it contains highly active molecules in low-to-moderate concentrations. Additionally, we have identified several leads that exceed the stated IC50 value: Chamarhodos sabulosa IC50 0.4-1.4 ug/ml, Swida sanguinea IC50 ~0.46 ug/ml, Sedum hybridum IC50 ~0.37 ug/ml, Oenotera biennia IC50 ~0.7 ul/ml (see [link]). We have to emphasize that assuming uniform distribution and maximum oral bioavailability these values would correspond to a mere ~30-40 ug/70 kg body weight drug dose, which is a very small quantity. Even with 1% bioavailability, the calculated dose will lead to sufficient blood concentrations after taking in few milligrams. Bear in mind, these estimations, just like all screening models, contain approximations aimed at increasing throughput.

@bowtiedshrike

It is unclear what quality this ‘plant extract library’ is. Have these extracts been validated for cytotoxicity?

Cytotoxicity assays are included in the screening protocols for whole extracts and isolated compounds. This is exemplified by the cytotoxicity evaluation protocol for lead extracts in HepG2 cell line provided above. Currently, we’re conducting MTT assays for antiglycation leads using HepG2 cells and primary peritoneal macrophages.

@bowtiedshrike

Are they water soluble?

During sample preparation, we make extract stock solutions in binary alcohol-water solvents, in which all extracts are completely soluble in non-physiologically high concentrations. At screening stages, all screens are done in aqueous buffer solutions starting with 5 ug/ml concentration, which is too physiologically high. All extracts were soluble in these aqueous solvents. Polymerization and extract osmolation might contribute to decrease in solubility during drying; we, however, work with liquid extracts.

@bowtiedshrike

Do they have a realistic chance of being stable/delivered even if they work in the functional assays?

Functionally and structurally, polyphenols analogous to commonly extracted components display sufficient stability, some of them (for instance, glycosidic forms) possess sufficient oral bioavailability. We have devised a methodology for enhanced complex analysis of compounds for their antiglycative properties in vivo: mice are intoxicated with methylglyoxal for 2 weeks (17.25 mg/kg intraperitoneally) while concomitantly fed a glycation inhibitor (the approach is calibrated using a known antiglycation compound ALT-711 – alagebrium). Subsequently, the mice are euthanized, liver and serum are collected for AGE content quantification. The model has proven successful: ALT-711 (50 mg/kg orally) completely abrogated methylglyoxal-mediated glycation. This technique permits simultaneous determination of the stability of compounds under study in biological media as well as their bioavailability. Our lead extracts will be tested using this methodology shortly.

@bowtiedshrike

It’s not clear from the budget how many people of what qualifications are working on the project

Our team consists of 3 PhDs, including 1 MD; 2 PhD students, who had to pause their degree acquisition process due to the ongoing war in Ukraine (at present, these team members are relocating to safer regions of the world). We also have 3 undergraduate students. In addition to the scientific collective, the team comprises 2 managers and 1 business lead.

@bowtiedshrike

or if it’s all out-sourced, or supply costs.

Some part of the research will indeed be outsourced. However, primary screening phases are set up and will be conducted by the scientific team.

@bowtiedshrike

If they want $25-$40k to prepare the extracts, they don’t have the extract library in hand.

This statement is incorrect. The indicated sum is dedicated to extract preparation (2500 in total which is a lot) from available raw material and initial chromatography profiling.

@bowtiedshrike

Why is $15k going to a video documentary?

Making documentaries and popular science videos are one of the means to promote the idea of radical life extension. Swaying public opinion towards accepting the idea of extending lifespan is one of the aspects of Open Longevity’s mission. Otherwise, VitaDAO and other communities oriented toward human longevity will inevitably face the problem of continuously searching for surrogate indications instead of combating aging as a process.

@bowtiedshrike

I thought patenting in the US and Europe ran closer to $30k than $10-$15k. Also, small molecules also have shorter patent times in the US now due to the Inflation Reduction Act. This will reduce the value of any IP generated around these molecules even if successful.

At this stage, we’re relying on the VitaDAO expert community’s help. However, if you recommend increasing the proposed amount, we’ll take your opinion into consideration. We believe we have enough evidence to hope for a successful realization of the project.

On behalf of Open Longevity and the Boston Matrix team we wish to thank you for your interest in our proposal and your critique! Critical evaluation of the project makes it stronger.

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I will incorporate their follow-up in the proposal, as well as update it with details on the surrogate indication when the text is ready (within a day or two). Thank you for your feedback!

[bowtiedshrike]

That reply clarified a lot about this proposal. It’s encouraging to see there is an assay in hand, and they have some leads.

However, I’m concerned about the assay. In the sample assay in the google doc with tannin, the “protection” comes from an increased background level, not a decrease in absorbance. This runs the risk of being a false positive. The ELISA is concerning because it has a low dynamic range (the max absorbance is 0.1). And it’s not clear if the ELISA is linear within that range. I would also note that most IC50s were extrapolated, which increases the error in the assay. Properly, the IC50s for most are <4.8 ug/mL. A second assay that is more robust seems needed to validate hits and confirm estimated IC50s.

The MTT assay has low precision, which is concerning. I do not think a ‘cytovitalic’ action can be concluded here. Instead, I would interpret negative numbers to indicate that extracts interfere with the assay. That means you’ll need a different assay to validate cytotoxicity. LDH is a terrible viability assay, so would recommend against that one. You might be able to do calcein release in a plate format, though.

@rpill

We’ve outlined our stage 2 working plan in the proposal…The markers can be divided into two groups: 1. linked to aseptic inflammation and inflammaging (pathogenically associated with aging [1] as well as with diabetic cardiomyopathy [2], and elicited by AGEs)

This still looks unfocused to me. There are many things that elevate cytokines, and the contribution of AGEs in the ECM to inflammaging and other phenotypes is questionable. These end up being different questions than ‘how do these plant extracts interfere with AGE accumulation?’ and measure potential downstream effects. Since RAGE is a therapeutic target, checking impacts on RAGE seems higher value to me than some of the others.

@rpill

range of mechanisms, such as protein amino group protection, transglycation, antioxidant action, amadorin activity, dicarbonyl scavenging, chelation of transition metal cations, etc.

This is where I would expect mechanistic studies to start, and then get more physiologic.

@rpill

Currently, we’re conducting MTT assays for antiglycation leads using HepG2 cells and primary peritoneal macrophages.

Bone-marrow derived macrophages might give you better numbers for scaling up- you can expand them in GM-CSF for 21 days and freeze them down after day 7. Or is the purpose of using the peritoneal macrophages because you elicited them with peptone (which has AGEs in it)? If that’s the case, why not aged thioglycollate medium?

@rpill

The indicated sum is dedicated to extract preparation (2500 in total which is a lot) from available raw material and initial chromatography profiling

It may be a communication issue, but this sentence indicates to me that the library is not ready to go. The raw materials are available, but the extracts that will be used are not yet ready.

Based on the time calculations, it’s not clear if the screen will go in 384-well plates or not. With a dedicated tech, and careful strategy (screen high concentration on all of them first, and eliminate any that fail to show activity there), you might be able to cut the number of plates you use down.

@rpill

Swaying public opinion towards accepting the idea of extending lifespan

I’m unconvinced this is a problem. It seems like it would distract from the science. If VitaDAO wants to fund videos, that would be better done as a seperate initiative. If you were submitting to the US National Science Foundation, though, this might be a reasonable Broader Impact.

@rpill

At this stage, we’re relying on the VitaDAO expert community’s help.

I think $30k was what was quoted to me by my TTO for the cost of filing just in US and Europe. Additional countries cost more. However, others in VitaDAO have more experience on this end than I, and will know precise amounts. Maybe it was just cost for filing the provisional patent?

[rpill]

@MAC

What about donating blood, phlebotomy to dump old glycated junk?

Hello, @MAC! Partially getting rid of glycated blood proteins has its benefits for sure. Primarily, circulating AGEs act on their cognate receptors – RAGEs. These receptors belong to the immunoglobulin family and are dedicated to sensing so-called damage-associated molecular patterns (DAMPs). AGE-dependent activation of RAGEs stimulates oxidative stress and expression of pro-inflammatory markers, so discarding glycated blood components is undoubtedly useful. However, removing AGEs from the extracellular matrix is by far a more complex task, hence the present proposal. I hope I answered your question.

[rpill]

@bowtiedshrike Members of the BostonMatrix team would like to express their gratitude for your effort in reviewing their proposal. Currently, the team is looking forward to collecting community feedback and will respond and introduce amendments to the text in bulk.

[Egorova]

@bowtiedshrike

If VitaDAO wants to fund videos, that would be better done as a seperate initiative.

Separating popularization and research projects has always been a rule of some sort. We’ve tried it. By we I mean all of us as a field. And I wouldn’t say we succeeded much in shifting the mindset of people, in forming the vision of longevity research. And we do need that for the field to grow, to grow fast enough for us to see the results of our work (we are not arguing about that, right?). The old ways of popularization, of promotion, of explanation don’t really work. We haven’t found the right format (among other reasons, it’s actually not that easy topic to cover in one paragraph, we do need to look, to experiment in the life-extension-popularization field too).

So what we propose is the new format: unite the actual research and a movie about it. Make it real. Show people how REAL it is. It’s not somewhere behind close doors, it’s very transparent, and anyone is welcome to join and help. Most people can only guess how the research itself looks like, what are the day-to-day obstacles, routine practices, how does the result and the intermediate result look like and thus, what is the possible pace of research.

I believe VitaDAO was created to build an active community. One tool for it is to publicly post scientific proposals and then publicly discuss them. Exactly what we are doing right now. But here we are: only 6 people voted and 4 people joined the discussion. The very discussion that will affect the protocol of the experiment and its chances to be executed at all. This exact discussion and these discussions in the field as a whole need scaling. And for this we need new formats, new ways of getting people engaged. That’s why the movie would be an important part of research.

[bowtiedshrike]

@Egorova

in forming the vision of longevity research. And we do need that for the field to grow, to grow fast enough for us to see the results of our work

I have mixed feelings on popularization in general. I would say popularization needs to be targeted, with a measurable effect, and that researchers tend to be the worst at marketing and sales. Selling extended life to the boomers ruling most countries and controlling governmental purse strings should be closer to selling heroin to junkies than ice to Alaskans.

If the plan is to do marketing, we need a clearly defined marketing campaign and budget. I suspect there are many Bowties that would do a great job developing resources if the pay was right. VitaDAO has also been active with media creation, so may not need outside help.

If the need is more people doing proposal review, I don’t think you’ll get what you want from a fancy video. Most lay people lack the expertise to evaluate the proposals, and will trust experts they otherwise trust. Some would rather read the discussion and use that to inform their decision, even if they don’t have anything to add.

Targeted recruitment for early career faculty or TTO personnel (and $Vita) would help get outside reviews on the proposals. That’s a cold email campaign, though, not a video campaign.

The other big challenge for decentralized organizations is time. Poll the VitaDAO membership and see how much time they are willing to invest in VitaDAO, between reading posts to stay up to speed, to evaluating different things. Each of the project proposals takes an hour or more to read, digest, and evaluate.

With a few exceptions, the most engaged people are already in Working Groups, where these ideas received a round of evaluation already. Most of them do not comment except to clarify, presumably because they don’t want to bias the community’s evaluation.

[Egorova]

Though we are in the VitaDAO space right now and I did a reference about our current conversation and campaign, this was just a mere example of what’s happening in the field in general. I actually don’t expect more interest and expertise from within the existing community and don’t expect the movie to work for that cause. We are all stretched pretty thin already. The question is how to grow the field in general.

We’re in desperate need of exponential growth of fundamental research. Meaning governmental funding. Meaning a massive shift in distribution of budgets. Meaning life extension has to win as an idea above any other. Win over the public, win over the people in power. Also, not only should we talk about “life extension is good”—this reasoning doesn’t seem to be working—but also about New Normal. Spending your lifetime trying to conquer death is the new normal and the only reasonable thing to do. Anything else is not cool. How does a life of such person look like? Here, the movie will show you (and hopefully many other movies and other engaging and immersive media will follow). It just seems like a waste not to put on camera the people who are already doing what everyone else is supposed to be doing with their lives (not being researchers necessarily but being part of the movement).

And yes, I absolutely agree that we need a measurement system, a KPI for popularization. And I suggest to work out an index for the life extension field in general: does it grow or not? Does the current fuss serves our goal of life extension? Do our efforts in enlarging the field work? Should we be optimistic about the current state of things or not? Different specialists in the field vary in their estimations, and all of them rely on their intuition. But I don’t want for it to be a competition of “I told you so” (which it comes down to eventually). I need to know properly in advance. I need the metric—our lives are at stake here. The metric could be a combination of different indexes or something fun like The Big Mac Index. A number of startups plus the budget of Buck Institute plus the number of TV shows, which portray radical life extension positively plus a number of politicians standing for the growth of research budgets plus… I’m just randomly naming things here, obviously we can’t just summarize it all. But I hope you get my point.

So my proposition (not within this research proposal but since we’re discussing this) is to work out the index for the field, create content on the new ethics, and see how one effects the other.

Going back to the video content: people tend to believe what they understand. And visa versa. If they don’t understand how life extension research work, they ignore it. If they see it and it looks doable, it inspires them to take part, to sponsor, to start their own research, to learn more. And the video made from within is just more sincere and more accurate. And yes, of course, not the researchers themselves are to be making the production—there are specialists to hire (director, editor, camera people…). It’s just we should be in control of the end message here.

[rpill]

@everyone
The VDP has been updated with details on diabetic cardiomyopathy as a surrogate indication and expanded on the methodology. Since the text has been changed considerably, votes have been reset. Please vote again if you think the the project should be funded with the requested amount.

As had been discussed at longevity dealflow working group meetings and in the designated thread in the WG Discord channel, in order to critically evaluate the project’s feasibility and attractiveness in terms of fundability, the applicant team has been asked to show proof-of-concept (PoC) efficacy of extracts in an in vivo model. Consequently, the team is asking $30k (see Experimental plan and Financing and milestones sections) to conduct a short PoC study in an experimental glycation animal model.

[rpill]

Hello, @bowtiedshrike! Please see below the applicant team’s response to your comments:

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@bowtiedshrike

In the sample assay in the google doc with tannin, the “protection” comes from an increased background level, not a decrease in absorbance.

Protection might seem to derive from an increased background level. However, the direct correlation between the background value and tannin concentration in non-glycated samples indicates the reliability of the result. There is no such relationship for glycated samples. Perhaps 100 uM of tannin (high concentration) disturbs the spatial organization of the protein, contributing to the nonspecific adhesion of labeled antibodies. We believe that blank subtractions are a reliable way to filter out this artifact.

@bowtiedshrike

The ELISA is concerning because it has a low dynamic range (the max absorbance is 0.1). And it’s not clear if the ELISA is linear within that range.

The discussed ELISA protocol is preliminary and will be validated further. ELISA parameters may be revised to improve data quality.

@bowtiedshrike

I would also note that most IC50s were extrapolated, which increases the error in the assay. Properly, the IC50s for most are <4.8 ug/mL. A second assay that is more robust seems needed to validate hits and confirm estimated IC50s.

A repeat study will be performed. Initial screening of extracts is conducted in a concentration range of 48-4.8 ug/ml, after which the investigated concentrations are reduced to 0.48 ug/ml (or lower) for potential lead extracts. The range of investigated concentrations in primary screening can exceed two orders of magnitude.

@bowtiedshrike

The MTT assay has low precision, which is concerning.

The low accuracy of the results for the extracts that exhibited a controversial “cytovitalic” effect may be related to the optical density values exceeding the linearity range of the test system. At the same time, for quercetin, which expressed cytotoxicity, the variability was within the acceptable range.

@bowtiedshrike

That means you’ll need a different assay to validate cytotoxicity. LDH is a terrible viability assay, so would recommend against that one. You might be able to do calcein release in a plate format, though.

To determine potential cytotoxicity in vitro, we usually use a combined approach that includes an assessment of the effects on cell growth and metabolic function:

1. assessment of morphological changes (Diff-Quik staining).
2. assessment of cytoplasmic membrane integrity (LDH test, trypan blue test, we also consider calcein test that you have recommended).
3. evaluation of metabolic functions of the cell (MTT-test in two modifications: under microscopy control and by spectrophotometry)

The above protocols aim to detect physiological and/or morphological changes that may have occurred as a result of cytotoxic action.

@bowtiedshrike

I do not think a ‘cytovitalic’ action can be concluded here. Instead, I would interpret negative numbers to indicate that extracts interfere with the assay.

The conclusion about the “cytovitalic” effect is controversial and requires verification. However, it is unlikely that the result is due to interference from the extracts. We remove the medium containing the extract and, in some cases, perform a series of cell washings before introducing the MTT reagent. To verify the result, we are going to perform a modification of the MTT test with microscopy detection. This implies mandatory microscopic control of formazan crystal formation, and only samples with intracellular crystal localization will be selected for further spectrophotometric evaluation, which will exclude the possibility of nonspecific interaction of the extracts under study with tetrazolium salts in the extracellular space.

In case of confirmation, we will consider the result as a certain qualitative sign indicating a possible “cytovitalic” effect of the extract components, which, however, will require further study of the mechanisms of the effect. For some plant polyphenols (ellagitannins, urolithin A), the ability to enhance mitochondrial activity and stimulate mitophagy was previously shown [link]. We cannot rule out a similar effect from some components of our extracts.

@bowtiedshrike

This still looks unfocused to me. There are many things that elevate cytokines, and the contribution of AGEs in the ECM to inflammaging and other phenotypes is questionable. These end up being different questions than ‘how do these plant extracts interfere with AGE accumulation?’ and measure potential downstream effects. Since RAGE is a therapeutic target, checking impacts on RAGE seems higher value to me than some of the others.

Many factors contribute to cytokine levels, but according to our understanding, AGEs, as well as matrix stiffness contribute significantly to the inflammatory phenotype through pattern recognition receptors [link], Hippo pathway [link], and other mechanisms. The view that AGEs and inflammation are linked is supported not only in scientific publications, but also in various projects [link]. Some interleukins are considered as markers of inflammation (e.g. IL-6 [link]), and causality of AGEs for markers of inflammation (e.g. IL-6 and IL-8 [link]) is described. Thus, we plan to evaluate the ability of our agents to suppress the AGE-induced inflammatory cell response (regardless of the detailed mechanism of the effect). One of the mechanisms of such action may be RAGE blockade. We recognize the importance of studying RAGE for understanding the mechanism of action of the agents, and are considering including it in the plan. However, the original exclusion of RAGE was because RAGE is a target whose development may require reevaluation of the financial plan. The question of including RAGE is not a question of whether or not we will include it in the study design, but it is a question of at what point in the research we will include it. However, we note that isolated blockade of RAGE may not be sufficiently effective (e.g., azeliragon clinical trials link).

The idea of reducing matrix stiffness through an antiglycation action is based on the fact that we prevent the glycation of proteins that have a long (but not super long) turnover period. The turnover period of such proteins is sufficient for formation of AGEs and cross-links, but it is not counted in years or decades. Such proteins are usually found in the structures of the pericellular matrix – laminin, type IV collagen, etc. (their turnover periods are as follows [link]). If these proteins are protected from glycation, the AGE load of their new generations will be lower, and therefore the associated stiffness and DAMP-potential less pronounced. The rate of matrix stiffness reduction, like the rate of matrix clearance from AGEs, will be proportional to the turnover period. This approach can be regarded as independent, but due to its partial nature, we consider it as a step in a complex therapy to restore tissues from the effects of glycation and aging. Subsequent steps, the implementation of which is difficult without reducing the AGEs load in ECM, include restoration of the synthetic activity of resident matrix cells, restoration of the stem cell population and their niches, senolysis, and stimulation of the turnover of extra-long-lived proteins. All named steps are considered by us as part of our master strategy [link].

We can offer another marker of matrix stiffness reduction that is promising specifically for plant polyphenols. Plant polyphenols can inhibit hyaluronidase [link], thereby slowing down the breakdown of hyaluronan. Hyaluronidase inhibition kits are available [link]. What do you think about including this marker in our application?

@bowtiedshrike

Bone-marrow derived macrophages might give you better numbers for scaling up- you can expand them in GM-CSF for 21 days and freeze them down after day 7. Or is the purpose of using the peritoneal macrophages because you elicited them with peptone (which has AGEs in it)? If that’s the case, why not aged thioglycollate medium?

The choice of peptone was based on reliability, simplicity, and in order to save costs. At the same time, if we are able to secure additional funding, we can change the experimental protocols, in particular consider the use of bone marrow-derived macrophages, use other activators and colony-stimulating factors.

At the planning stage of the study, we did not consider the connection between the mechanism of macrophage activation by peptone and the presence of AGE in peptone, but we will consider the potential significance of such a connection in future work. The first step will be to investigate the peptone used for the presence of nontryptophan fluorescence (fluorescent AGEs) in it. Perhaps its role will prove to be significant.

@bowtiedshrike

This is where I would expect mechanistic studies to start, and then get more physiologic.

We agree. This is the way we organize our project.

@bowtiedshrike

It may be a communication issue, but this sentence indicates to me that the library is not ready to go. The raw materials are available, but the extracts that will be used are not yet ready.

The raw material is ready for the extraction procedure, but the extracts are prepared in sequence as soon as we are ready to take a new batch into the screening. The extraction procedure takes 24 hours, and it takes some time to determine the concentration of the extract. Thus, the extraction procedure is not a time-limiting stage of the project. There is enough raw material in stock to prepare quantities of the extract sufficient for brief in vivo studies for lead candidates. In addition, there is the possibility of determining plant ITS markers for clear species identification if more raw material is needed. These factors contribute to extraction costs.

@bowtiedshrike

Based on the time calculations, it’s not clear if the screen will go in 384-well plates or not. With a dedicated tech, and careful strategy (screen high concentration on all of them first, and eliminate any that fail to show activity there), you might be able to cut the number of plates you use down.

During the screening, we use 96-well plates, although we can technically increase the number of wells to 384. The plan to examine all extracts for activity over ~5-month period is not based on a predictive calculation, but on real estimates of the throughput of the test system we had performed earlier.

@bowtiedshrike

I think $30k was what was quoted to me by my TTO for the cost of filing just in US and Europe. Additional countries cost more. However, others in VitaDAO have more experience on this end than I, and will know precise amounts. Maybe it was just cost for filing the provisional patent?

We would appreciate to have your detailed feedback on what costs we might incur and how to put them in an optimal sequence, as patenting is not our strongest side.

On behalf of OpenLongevity and the BostonMatrix Team, we thank you for your questions.

We will continue to collect feedback from the VitaDaO community to improve our project further.

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[scienceman]

Hello BM team - can you clarify per stage 3 if the extract structures are not amendable to synthesis you propose to move to medical chemistry to explore a traditional small mol approach?

As we discussed live, in general I am not a fan of natural products. However this project becomes interesting if at minimum we gain understanding of MOA which can be used to launch discovery using standard drug like modalities. At best, one of the extract components can be synthesized easily & offers an immediate path forward.

Also, I do not understand the need for a promotional video….

[RomanLitvinov]

Hello, thank you for the interest to our proposal!

@scienceman

can you clarify per stage 3 if the extract structures are not amendable to synthesis you propose to move to medical chemistry to explore a traditional small mol approach?

If the first-best compound is not amendable to synthesis, we will select second best, third best etc. We plan to assess 2500 extracts, which increases the chance of finding several leads. However, we agree that it is sensible to add this point into “Risks” section of this application.

Should all lead compounds not be amendable to synthesis, it is possible to use the lead structures as the blueprint for synthesizable drug-like molecules. We can narrow the pool of predicted drug-like molecules by testing them for antiglycation activity using our proprietary in silico methods [published here] and by in silico ADME-Tox screening. This approach will result in a novel synthesizable drug-like component with properties close to the nature-derived lead.

We also note that there is a chance that the lead can be synthesized at scale by biocatalysis. There are known cases of applying this approach to synthesize several flavonoids [1, 2].

@scienceman

As we discussed live, in general I am not a fan of natural products. However this project becomes interesting if at minimum we gain understanding of MOA which can be used to launch discovery using standard drug like modalities. At best, one of the extract components can be synthesized easily & offers an immediate path forward.

We agree that there is a high chance of finding at least one lead. We planned to uncover MoA of lead compounds in glycation reaction (transglycation, carbonyl compound binding, antiradical action, binding of transition metals etc). These trials are planned at Stage 1 (for extracts) and Stage 3 (for isolated compounds) of this application. In addition, we will conduct phenotypical screening to assess the compound’s propensity to suppress glycation-related pathological processes. Moreover, we are interested in further exploring MoA of our lead compounds.

As we proposed in our follow up [link] to live discussion, after identification of the active compound it can be immobilized on resin or magnetic beads and incubated with homogenized tissues. Further, captured molecules will be subjected to mass spectrometry and chromatography. These kinds of experiments are promising and results might be very interesting but it will require additional funding. We would expect to see such targets as kinases of NFkB or MAPK pathways, as these were previously identified to be involved in AGEs-associated pathogenesis, and also we hope to see GLO, PARK7, or AKR enzymes as endogenous glycation suppressors.

The establishment of detailed mechanisms of antiglycation activity represents considerable interest for us, and we are interested in identification of other targets as well. This justifies multiple tests for different types of antiglycation activity that we consider to qualify as a detailed analysis of the mechanism of action.

Thanks a lot for your questions, and have a great day.

Roman Litvinov, BostonMatrix Research Group

[Egorova]

Hi! Well, if the movie idea continues to raise questions, we’re not insisting on it within this fundraising campaign and can drop it. But we still like it and still would like to pursue with it (we’ll just have to look for the funding for the movie elsewhere). As to why, to summarize my longer previous answer, I think we have to use any given chance to expand the field in general, and making the movie about the research helps promote it and explains its inside to the general public. Personally, I would love to see the movies about all the ongoing research in the field: to meet the teams, understand their reasoning better, see the designs and the routines of the experiments explained, understand the difficulties, the struggles, how much does and doesn’t rely on human factor and on the accessibility to resources, tools, and logistics, to estimate the chances of success… I really think that all this will help with funding for the field in general. But again, we are not pushing.

[timrpeterson]

Sorry if this has been asked elsewhere but I’d like to see funding the 3rd and possibly 2nd milestones contingent on other funding sources coming in. $300k is becoming more than vitadao will typically invest. Also it is important to show traction with multiple funders.

[strygah]

We would definitely need to find a lot more additional funding for the project to succeed and are looking for it already (in parallel to VitaDAO). It is though quite hard, given the very early stage of the project. We would be OK with contingent funding for a 3rd milestone. Would something like a combination of charity, gitcoin, other crowdfunding, private funding and/or grants suffice or are you looking for something more specific? What sum shall we agree upon?