VDP-[] Boston Matrix

One-liner: Discovery and development of novel antiglycation and anticross-linking 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

Scientific evaluation: TBA
Business evaluation: TBA

Shepherd: Rakhan Aimbetov
Other squad members: Estefano Pinilla, Paolo Binetti, Max Unfried, Laurence Ion

Sourced by: Rakhan Aimbetov

Project PI: Dr. Roman Litvinov

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 leading to the loss of ECM elasticity and stiffness. Several researchers consider ECM stiffness as a new hallmark of aging that drives cellular senescence, stem cell exhaustion, and chronic systemic inflammation.

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.

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. 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. 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 very long half-lives.

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, – or using chemical compounds like ALT-711. 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 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.

Opportunity

The Boston Matrix team is planning to screen over 2500 natural plant extracts, isolate and develop a novel therapeutic candidate. The mechanism of action 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 (1000+ over ~7 years).

The team has access to a library of 2500+ natural extracts (and means to identify, isolate and modify active ingredients to make patentable products with drug-like properties) and to 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 tools are based on 199 molecules and natural extracts (most data is unpublished) with a proven antiglycation effect.

Most natural plant extracts contain polyphenols, flavonoids, anthocyanidins, and other active compounds. Preliminary trials indicate that quercetin, luteolin, and tannin have shown 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). This and the whole history of pharmacology indicates 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 modelling pathology (in this case – the glycation process) for initial screening. After that, the leading candidates will be tested in cellulo. They do not plan animal studies at this stage as they are not able to fit the trials in a 12-months timeframe and in the budget.

Potential non-aging indications for regulatory approval are type 2 diabetes and its long-term complications (nephropathy, angiopathy, etc.), 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 by some researchers). Other possible applications of the platform with faster time-to-market are the cosmetic dermatology industry, cosmeceuticals, and nutraceuticals.

Some of the indicated results obtained by the scientific groups, which included Roman Litvinov, were published in peer-reviewed journals. Some examples are:

https://doi.org/10.1016/j.ejmech.2019.111808

Experimental plan

Stage 1:

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

The purpose of this stage is to assess the antiglycation activity of ~2500 natural extracts rapidly and cost-effectively in an assay modelling accelerated glycation. The team will be monitoring glycation end products (for all extracts) and intermediate products of glycation (for lead extracts). Progressively, as they are able to weed out more extracts, they will make the assay more complex by introducing additional parameters (temperature, pH, ion composition) and using ELISA. This is aimed at improving the specificity of the results. Cytotoxicity studies of the most promising candidates are also planned for this stage.

Stage 2:

  • Short-listing and further analysis of lead natural extracts. Initial talks with patent attorneys. [additional 2-4 months after the end of stage 1]:
  1. additional assessment of antiglycation activity with ELISA
  2. exploration of antiglycation activity mechanisms

Promising candidate extracts from stage 1 will be tested in cellular models. The team plans to test a relatively narrow range of markers that change due to glycation. The long-listed markers are cellular AGEs levels, inflammatory cytokines levels (IL1b, IL6, IL8, TGFb, and other), metalloproteinase levels (MMP2, MMP9, TIMP1, and other), distinct cellular phenotypic changes (fibroblast migration and other) or differentiation changes of stem cells. The final choice of markers will depend on the results of the first experiments. Part of these trials is planned to be outsourced.

Stage 3:

  • Testing the activity of isolated active compounds (3-4 months after stage 2):
  3. preparative chromatography
  4. additional exploration of antiglycation activity including physical methods (s.a. dynamic PMR)
  5. exploration of complementary activities (RAGE inhibition, anticrosslinking, matrix stiffness tests)
  6. cytotoxic screening
  • Optimizing the lead candidate structure via an AI-powered tool and structural biology 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 of 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 in repeating stages 1-2.

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-products (AGEs) inhibitors, Cross-link 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.

Nikita Valuysky, researcher.

Marina Gasheva, researcher.

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 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 $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: $145k
Milestone:

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

Second financing stage: $155k
Milestones:

  • One 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 2 milestones the overall timeline might increase, as some processes that might have been done in parallel would be performed sequentially.

Strengths

  • The group possesses unique experience in a relatively new field – glycation reaction and cross-links as a target for longevity interventions.
  • Their preliminary PoC data show a promising antiglycation effect of several natural plant-derived compounds.
  • IC50 of these compounds is two orders of magnitude lower than the reference antiglycation compound aminoguanidine 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 inhibition of glycation could have no effect on already glycated macromolecules, therefore only delaying damage accumulation rather than repairing *.
  • 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).

* 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.

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