2024-05-30

Q: What is Telomir-1, and how does it work to lengthen DNA's telomere caps?

Dr. Chris Chapman: Telomir-1 is a new chemical entity based on a synthetic alkaloid. This alkaloid turns on telomerase in human stem cell cultures.

In collaboration with Frontage Laboratories, a study was conducted for telomere length quantification. Briefly, MRC-5, HUVEC, or MSCs were grown to confluency and plated in a 12-well plate either untreated or treated with the Telomir-1 compound. After 48 hours, DNA was extracted and then DNAs were subjected to qPCR via a kit. To evaluate our hypothesis that Telomir-1 influences telomere length, we conducted tests on human primary cell strains, specifically HUVEC, MRC-5, and Bone Marrow-Derived Mesenchymal Stem Cells. These particular strains were chosen due to their known propensity for senescence through cell passaging. Telomir-1 was evaluated for 1) cell cytotoxicity, 2) Telomere length modulation, 3) cell proliferation, and 4) Telomir-1-induced cell death.

Telomir-1-mediated cytotoxicity was assessed. MRC-5, HUVEC, and MSCs were treated with Telomir-1 at 1, 10, 25, 50, 100, 250, 500, and 1000 µM. Telomir-1 demonstrates no cytotoxicity at 1-250 µM concentration in MRC-5 (Figure A) and HUVEC (Figure B), while at 500 µM, a major cell loss was observed. In MSCs, cell loss was detected in culture treated with a vehicle, while Telomir-1 augmented the cell loss (Figure C). Therefore, MRC-5 strains and Telomir-1 compounds at 1-500 µM concentrations were chosen for subsequent studies.

Telomir-1 cytotoxicity results and telomere length qPCR results. MRC-5, HUVEC, and MSC cells were treated with increasing concentrations Telomir-1 to demonstrate Telomir-1-induced cytotoxicity. 48-hour Telomir-1-treated MRC-5, HUVEC, and MSC cell DNA extracts were assayed by telomere length qPCR. Results were calculated based on calculations using a telomere primer set that recognizes and amplifies telomere sequences, and a single copy reference primer set that recognizes and amplifies a 100 bp-long region on human chromosome 17 as an internal control. EtOH-treated cells at 1% (equivalent to 1 mM Telomir-1 treatment) served as vehicle control in all experiments.

MRC-5, HUVEC, and MSCs were treated with Telomir-1 at 1, 50, 100, and 500 µM concentrations (based on cytotoxicity data) and assessed for telomere length using qPCR. Telomere length was calculated as total telomere length/ diploid cell. A trend where Telomir-1 induced telomere lengthening could be observed at concentrations greater than 1 µM in all three cell lines, with some of the largest lengthening effects seen at 50 µM. Based on this observation, it suggests that the effect of the Telomir-1 compound is not cell-type restrictive.

MRC-5 was treated with Telomir-1 at 500 µM, a known concentration that led to cell loss in MRC-5. The treated cultures were then triple-stained with an anti-active Caspase-3 antibody, dead cell exclusion dye, and FITC-conjugated Annexin V, followed by flow cytometric analysis. Telomir-1 induced cell death in MRC-5, as evidenced by highlighted staining of Scarlet-conjugated dead cells and FITC-conjugated Annexin V channels. The double-positive cells were further analyzed for active Caspase-3 activity, where the population was considered apoptotic rather than necrotic. The heightened Caspase-3 activity indicates that the cells are going through an apoptotic cell death pathway, in contrast to necrosis.

Telomerase Activity Assessment

MRC-5 Passage 4 (P4) cells were treated with 1, 50, 100, and 500 µM of Telomir-1. Cells were then lysed and processed for telomerase activity using qPCR. We found that Telomir-1 enhanced Telomerase activity in MRC-5 cells in a dose-dependent manner (~40%). The enhancement of telomerase activity ceased to be effective at 500 µM, likely due to cell cytotoxicity.

Q: How does Telomir-1 help in maintaining cellular metal homeostasis and what diseases are targeted by Telomir-1 due to its metal binding properties?

Dr. Chris Chapman: Telomir-1 reversibly binds to essential metals preventing the ionization of those metals due to the Fenton reaction and therefore limiting their biological effects. Age-related diseases such as osteoarthritis, senile dementia, macular degeneration, hemochromatosis, and muscle wasting are just some of the potential applications. Although metal reactivity is required for eukaryotic organisms to reach healthy maturity, continued elevated and unregulated metal-induced enzymatic activity contributes to oxidation and inflammation. It has long been recognized that enzymatic reactions also play a key role in the formation of cancerous mutations and the development of diseases like hemochromatosis. By limiting available metals, thus inhibiting metal-induced enzymatic activity, Telomir-1 may prove to be an effective platform for treating a variety of diseases.

Q: Could you describe the safety profile and potential side effects observed in the preclinical studies of Telomir-1?

Dr. Chris Chapman: We have completed several preclinical studies to date, including but not limited to the following:

• Plasma Protein Binding
• Reaction Phenotyping in Human Liver Microsomes
• Metabolite Identification
• Recombinant Enzymes (CYP450)
• Direct and Time-Dependent Inhibition of CYP450
• Maximum Tolerated Dose/7-Day Dose Range Finding in Rat
• Maximum Tolerated Dose/7-Day Dose Range Finding in Dog

We have observed no toxicity signals at concentrations that in cell cultures are effective doses. 98% of Telomir-1 is excreted in the urine unchanged.

Q: What future research directions are planned for Telomir-1 and can we expect its production on a global level, particularly concerning its application in human clinical trials?

Dr. Chris Chapman: Yes, studies on osteoarthritis in animals with the disease including dogs are planned; future human studies based on these animal results are planned as well. We have secured manufacturing commitments from global manufacturers.

In collaboration with Charles River Laboratories, we are currently conducting an osteoarthritis study in 30 adult Sprague Dawley rats ages 7-9 months.

In collaboration with ARGENTA – Midwest Veterinary Services, Inc., we are planning an osteoarthritis study that will include 10 canines greater than 2 years of age. Additionally, we will be conducting a safety study that will include 5 geriatric canines.

Telomir Pharmaceuticals will pursue both an animal IND and a human IND for osteoarthritis. We are considering additional age-related diseases with guidance from the FDA.

Q: How could potential therapies like Telomir-1 impact the management of diseases related to telomere shortening and metal overload?

Dr. Chris Chapman: Telomir-1 is the only drug we know of that has been shown in human cell stem cell culture to substantially increase Telomerase and thereby increase the length of telomeres. We believe this 10X increase in telomeres explains its unprecedented ability to reverse aging and age-related diseases.