Epitalon Research & Studies

Diabetic retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of vision loss. Short peptides, such as di-, tri-, and tetrapeptides, have various beneficial activities, including antioxidant, antimicrobial, and anti-inflammatory effects. This study aims to test the hypothesis that the antioxidant effect of the synthetic tetrapeptide AEDG (Ala-Glu-Asp-Gly, Epitalon) improves the delayed healing process associated with hyperglycemia in DR, using a high glucose (HG)-injured human retinal pigment epithelial cell line (ARPE-19). We found that HG exposure delayed wound healing in ARPE-19 cells and increased intracellular levels of reactive oxygen species (ROS), while decreasing antioxidant gene expression. HG also induced epithelial-mesenchymal transition (EMT) and upregulated fibrosis-related genes, suggesting that HG-induced EMT contributes to subretinal fibrosis, the end-stage of eye diseases, including proliferative DR. The antioxidant Epitalon restored impaired wound healing in HG-injured ARPE-19 cells by inhibiting hyperglycemia-induced EMT and fibrosis. These findings support using the antioxidant agent Epitalon as a promising therapeutic strategy for DR to improve retinal wound healing compromised by hyperglycemia. More mechanistic investigations are needed to confirm Epitalon's benefits and safety. Developing ophthalmic forms of Epitalon may enhance its delivery directly to the retina, potentially improving its therapeutic efficacy.

Epitalon, a naturally occurring tetrapeptide, is known for its anti-aging effects on mammalian cells. This happens through the induction of telomerase enzyme activity, resulting in the extension of telomere length. A strong link exists between telomere length and aging-related diseases. Therefore, telomeres are considered to be one of the biomarkers of aging, and increasing or maintaining telomere length may contribute to healthy aging and longevity. Epitalon has been the subject of several anti-aging studies however, quantitative data on the biomolecular pathway leading to telomere length increase, hTERT mRNA expression, telomerase enzyme activity, and ALT activation have not been extensively studied in different cell types. In this article, the breast cancer cell lines 21NT, BT474, and normal epithelial and fibroblast cells were treated with epitalon then DNA, RNA, and proteins were extracted. qPCR and Immunofluorescence analysis demonstrated dose-dependent telomere length extension in normal cells through hTERT and telomerase upregulation. In cancer cells, significant telomere length extension also occurred through ALT (Alternative Lengthening of Telomeres) activation. Only a minor increase in ALT activity was observed in Normal cells, thereby showing that it was specific to cancer cells. Our data suggests that epitalon can extend telomere length in normal healthy mammalian cells through the upregulation of hTERT mRNA expression and telomerase enzyme activity.

Epitalon, also known as Epithalon or Epithalone, is a tetrapeptide, Ala-Glu-Asp-Gly (AEDG), which was synthesized based on the amino acids composition of Epithalamin, a bovine pineal gland extract, prior to its discovery in pineal gland polypeptide complex solution. During the last 25 years, this compound has been extensively studied using in vitro, in vivo, and in silico methods. The results of these studies indicate significant geroprotective and neuroendocrine effects of Epitalone, resulting from its antioxidant, neuro-protective, and antimutagenic effects, originating from both specific and nonspecific mechanisms. Although it has been demonstrated that Epitalon exerts, among other effects, a direct influence on melatonin synthesis, alters the mRNA levels of interleukin-2, modulates the mitogenic activity of murine thymocytes, and enhances the activity of various enzymes, including AChE, BuChE, and telomerase, it remains uncertain whether these are the sole mechanisms of action of this compound. Moreover, despite the considerable volume of research on the biological and pharmacodynamic characteristics of Epitalon, the quantity of physico-chemical and structural investigations of this peptide remains quite limited. This review aims to conclude the most important findings from such studies, thus presenting the current state of knowledge on Epitalon.

Telomerase is highly expressed in oocyte cumulus cells and plays a significant role in follicular development and oocyte maturation. In this study, we hypothesized that in vitro culture conditions may affect telomerase activity during in vitro embryo production (IVP) and that its activation may improve embryo quality. We first examined telomerase protein levels and localization in bovine cumulus-oocyte complexes via immunofluorescence assays. The results showed that healthy cumulus-oocyte complexes have the nuclear localization of the telomerase while the degraded cumulus-oocyte complex had reduced telomerase levels and that telomerase was localized in the cytoplasm. We activated telomerase via Epitalon, a tetrapeptide with the amino acid sequence Ala-Glut-Asp-Gly. We observed a significant improvement in the oocyte maturation rate compared with the control group (p < 0.05). Furthermore, telomerase activity was significantly compromised in post-thawed embryos, and Epitalon treatment significantly improved blastocyst hatching rate and implantation potential (p < 0.05). Moreover, we performed qPCR, reactive oxygen species, and JC-1 (ΔΨm) assays to evaluate the effect of Epitalon on the health of in vitro mature oocytes, cumulus cells, and post-thawed blastocysts, and the result showed that Epitalon highly enhances the quality and health of the oocyte, cumulus cell, and post-thawed blastocyst. Our results suggest that telomerase activation via Epitalon improves bovine in vitro embryo production.

It was shown that AEDG peptide (Ala-Glu-Asp-Gly, Epitalon) regulates the function of the pineal gland, the retina, and the brain. AEDG peptide increases longevity in animals and decreases experimental cancerogenesis. AEDG peptide induces neuronal cell differentiation in retinal and human periodontal ligament stem cells. The aim of the study was to investigate the influence of AEDG peptide on neurogenic differentiation gene expression and protein synthesis in human gingival mesenchymal stem cells, and to suggest the basis for the epigenetic mechanism of this process. AEDG peptide increased the synthesis of neurogenic differentiation markers: Nestin, GAP43, β Tubulin III, Doublecortin in hGMSCs. AEDG peptide increased Nestin, GAP43, β Tubulin III and Doublecortin mRNA expression by 1.6-1.8 times in hGMSCs. Molecular modelling method showed, that AEDG peptide preferably binds with H1/6 and H1/3 histones in His-Pro-Ser-Tyr-Met-Ala-His-Pro-Ala-Arg-Lys and Tyr-Arg-Lys-Thr-Gln sites, which interact with DNA. These results correspond to previous experimental data. AEDG peptide and histones H1/3, H1/6 binding may be one of the mechanisms which provides an increase of Nestin, GAP43, β Tubulin III, and Doublecortin neuronal differentiation gene transcription. AEDG peptide can epigenetically regulate neuronal differentiation gene expression and protein synthesis in human stem cells.

This report discusses the properties of the synthetic tetrapeptide epitalon (Ala-Glu-Asp-Gly), synthesized on the basis of an epiphyseal peptide extract. Intranasal administration of epitalon was selected as a noninvasive means of applying the agent to the CNS by bypassing the blood-brain barrier. The aim of the present work was to assess the characteristics of the action of epitalon on the frequency of spontaneous neuron activity in the cerebral cortex of white rats. Studies were performed using male Wistar rats anesthetized with urethane (1 g/kg). Extracellular activity of cortical neurons was recorded with a glass microelectrode of resistance 1-2 MOmega. Recording of spontaneous neuron discharges for 10-15 min was followed by intranasal administration of epitalon solution and recording of neuron activity to 30 min after doses of 30 ng per animal. Significant activation of neuron activity was seen several minutes after dosage, with an increase (by factors of 2-2.5) in discharge frequency. In some experiments, the effect of epitalon was multiphasic. The first peak of increased neuron discharge frequency at 5-7 min was followed by peaks at 11-12 and 17-18 min. The increase in discharge frequency occurred because of an increase in the discharge frequency of neurons which were already active and the recruitment of previously silent neurons. At least the first peak of increased neuron activity following exposure to epitalon was found to be associated with the direct action of the peptide on cortical cells.

The potential preventive effect of the synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on spontaneous tumorigenesis in mice was studied. One-year-old female C3H/He mice were kept for 6.5 months under standard conditions. Epitalon was injected at a dose of 0.1 microg, 5 times a week. Long-term exposure to Epitalon in small doses did not show any toxic effect. Treatment with Epitalon decreased the number of tumor-bearing mice with malignant tumors and prevented the development of metastases. Spontaneous tumors of the reproductive organs (mammary glands and ovaries) were predominant in both groups of mice (control and experimental). The mammary gland tumors were different variants of invasive ductal carcinomas. In the ovaries, granulosa-cell tumors were found. Tumors were in the minority in other organs and had benign characteristics. In control mice, metastases were found in 3 out of 9 tumor-bearing mice, all of them being from tumors of the reproductive organs. Treatment with Epitalon slowed down the development of metastases from spontaneous tumors, and no metastases were found in the experimental mice. These data highlight the antimetastatic effect of Epitalon as part of its oncostatic properties.

Female senescence accelerated mice SAMP-1. (prone) and SAMR-1 (resistant) were exposed 5 times a week monthly to melatonin (with drinking water 20mg/ml during the night hours) or to s.c. injections of epitalon (Ala-Glu-Asp-Gly) at a single dose 1mkg/mouse. Control mice were intact or exposed to injection of 0.1 ml normal saline. The body weight and temperature, food consumption, estrous function were monitored regularly. The life span and tumor incidence were evaluated as well. As age advanced, the weight increased whereas food consumption and body temperature did not change. There was no significant substrain difference in these parameters. Exposure to melatonin or epitalon also failed to influence those indices. As age advanced, the incidence of irregular estrous cycles increased both in SAMP-1 and SAMR-1, whereas the treatment with both melatonin and epitalon prevented such disturbances. SAMP-1 revealed some features of accelerated aging as compared to SAMR-1. The mean life span of the 10% of the last survivors among treated SAMP-1 was shorter than that of SAMR-1, aging rate increased and mortality doubling time decreased. There was a direct correlation between body mass of the two substrains at the age of 3 and 12 months matched by body mass increase and longer life span. Melatonin or epitalon treatment was followed by longer mean and maximum survival in the 10% of the last survivors among SAMP-1. Melatonin involved decreased aging rate and increased mortality doubling time. Malignant lymphomas predominated in SAM without any significant difference in frequency between the substrains. While melatonin failed to influence tumor incidence or term of detection in SAMP-1, neither did epitalon affect frequency. However, it was followed by longer survival in tumor-free animals. No link between melatonin or epitalon treatment, on the one hand, and carcinogenesis, on the other, was reported in SAMR-1.

The effect of the synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on proliferative activity in colon tumors, and in mucosal epithelial cells adjacent to and located far from tumors was studied in rats. To evaluate the effect of Epitalon on different stages of carcinogenesis, different treatment regimens were used: during the tumor initiation stage, during the tumor-promotion stage, or during the entire process of tumor development. Eighty 2-month-old male LIO rats were exposed weekly to five subcutaneous injections of 1,2-dimethylhydrazine (DMH) at a single dose of 21 mg/kg body weight. Rats were divided into four groups. Control rats (group 1) received saline at a dose of 0.1 ml during the entire experiment. Rats in group 2 were treated with Epitalon at a dose of 1 micro g, five times a week, for 6 months, from the first injection of DMH till the end of the experiment. Rats in group 3 were treated with Epitalon after termination of the carcinogen injections. Rats in group 4 were treated with Epitalon only during the period of DMH exposure (for the first 5 weeks of the experiment). DMH induced proliferation of the secretory epithelium, and this phenomenon was accompanied by a decrease in the size of the stromal area and the area of lymph infiltration in colon tumors and in the colon mucosa adjacent to the tumors (group 1). Epitalon attenuated this effect, especially when the treatment was continued throughout the experiment (group 2). It increased the stromal areas, as well as that of lymphoid infiltration in the colon mucosa adjacent to the tumors. The intensity of lymphoid infiltration was activated in both the colon mucosa adjacent to a tumor and in the tumor. Mitotic activity of tumor cells was significantly inhibited by Epitalon when the treatment was given throughout the experiment (group 2). In parallel, a high level of apoptosis was seen in the same group. Thus, the strongest inhibitory effect of Epitalon on carcinogenesis in the colon mucosa was manifested when the treatment was continued throughout the experiment.

From the age of 3 months until their natural deaths, female outbred Swiss-derived SHR mice were subcutaneously injected on 5 consecutive days every month with 0.1 ml of normal saline (control) or with 1.0 microg/mouse (approximately 30-40 microg/kg) of tetrapeptide Epitalon (Ala-Glu-Asp-Gly) dissolved in 0.1 ml saline. There were 54 mice in each group. The results of this study show that treatment with Epitalon did not influence food consumption, body weight or mean life span of mice. However, it slowed down the age-related switching-off of estrous function and decreased the frequency of chromosome aberrations in bone marrow cells (by 17.1%, P<0.05). It also increased by 13.3% the life span of the last 10% of the survivors (P<0.01) and by 12.3% the maximum life span in comparison with the control group. We also found that treatment with Epitalon did not influence total spontaneous tumor incidence, but inhibited the development of leukemia (6.0-fold), as compared with the control group. The data obtained suggest a geroprotector activity of Epitalon and the safety of its long-term administration in mice.

The content of C-Fos protein was tested in rat pinealocytes in the norm and stress and in case of intranasal administration of Epitalon (Ala-Glu-Asp-Gly), which regulated pineal secretion processes, presumably, via protooncogenes.

The effect of synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on colon carcinogenesis was firstly studied in rats. Eighty 2-month-old outbred male LIO rats were subdivided into four groups and were weekly exposed to five subcutaneous injections of 1,2-dimethylhydrazine (DMH) at a single dose of 21 mg/kg body weight. Additionally, 5 days a week, some of the rats were given subcutaneous injections of saline at a dose of 0.1 ml during the whole experiment (group 1, control) or Epitalon at a single dose of 1 microg during the whole experiment (group 2), Epitalon after termination of carcinogen injections (group 3) or during the period of DMH exposure (group 4). Colon carcinomas developed in 90-100% of DMH-treated rats. The number of total colon tumors per rat was 4.1; 2.7; 3.7; 2.9 in groups 1, 2, 3, 4, respectively (the difference in groups 2 and 4 compared with group 1 is significant). In rats from group 2, colon tumors were smaller than in control animals. In group 2, the incidence, as well the multiplicity of tumors in ascending and descending colon, were significantly decreased in comparison with group 1. In group 4, the mean number of tumors per rat was significantly decreased, too. A trend to decrease the number of tumors in the rectum in rats from groups 2, 3 and 4, treated with Epitalon was found. Epitalon inhibited also the development of tumors in jejunum and ileum. Thus, our results demonstrated an inhibitory effect of Epitalon on chemically induced bowel carcinogenesis in rats.

Female FVB/N HER-2/neu transgenic mice from the age of 2 months were subcutaneously injected with saline, the peptide Epitalon(R) (Ala-Glu-Asp-Gly) or with the peptide Vilon(R) (Lys-Glu) in a single dose of 1 microg/mouse for 5 consecutive days every month. Epitalon treatment reduced the cumulative number and the maximum size of tumors (p < 0.05). Furthermore, the number of mice bearing 1 mammary tumor was increased, whereas the number of mice bearing 2 or more mammary tumors was reduced in Epitalon-treated in comparison to saline-treated animals (p < 0.05). The size but not the number of lung metastases was reduced in Epitalon-treated compared to saline-treated mice (p < 0.05). The treatment with Vilon produced significant negative effects when compared to the control group, with an increased incidence of mammary cancer development (p < 0.05), a shorter mean latent period of tumors (p < 0.05) and an increased cumulative number of tumors (p < 0.05). A 3.7-fold reduction in the expression of HER-2/neu mRNA was found in mammary tumors from HER-2/neu transgenic mice treated with Epitalon compared to control animals. The expression of mRNA for HER-2/neu was also partially reduced in Vilon-treated mice, but it remained significantly higher in Vilon- than in Epitalon-treated animals (1.9-fold increase). The data demonstrate the inhibitory effect of Epitalon in the development of spontaneous mammary tumors in HER-2/neu mice, suggesting that a downregulation of HER-2/neu gene expression in mammary adenocarcinoma may be responsible, at least in part, for the antitumor effect of the peptide.

Female transgenic FVB mice transfected with the mammary erbB-2/neu oncogene were injected 0.1 ml 0.9% solution of sodium chloride (control), 1 meg Vilon peptide (Lys-Glu) or Epitalon peptide (Ala-Glu-Asp-Glu), s.c., 5 days in succession once a month, beginning from the age of 2 months. The characteristics of mammary tumor induction in the control and experimental groups did not differ until the age of 9 months. Later on, Epitalon-treated mice revealed distinct inhibition of carcinogenesis. One tumor per animal was detected in 7% (control), 4% (Vilon) and 16% (Epitalon) (p < 0.05). Two or more tumors per animal were in 75%, 95% and 56%, respectively (p < 0.05). Largest diameter of mammary adenocarcinoma in the Epitalon group was smaller than in controls by 33% (p < 0.05). Although the number of mice with metastases to the lung in all three groups was practically identical, their incidence in the Vilon group was 2.6 times higher than in Epitalon-treated animals (p < 0.05). Largest diameter of metastasis in the Epitalon group was the smallest, too. Our data point to inhibition of mammary carcinogenesis by Epitalon in transgenic erbB-2/neu mice.

We have studied the effect of tetrapeptide Epitalon (Ala-Glu-Asp-Gly) on the course of congenital pigmented degeneration of the retina. The application of Epitalon in Campbell rats is found to intensify the bioelectric and functional activity of the retina due to the preservation of its morphological structure. Epitalon therapy in patients with degenerative retinal lesions results in a positive clinical effect in 90% of the cases. The analysis of Epitalon effects suggests that the tetrapeptide participates in the mechanisms of transcription common for the epiphysis and retina.

The aim of this research consisted in studying the effects of tetrapeptide Epitalon on both thymocyte proliferation and interleukin-1beta (IL-1beta) signal transduction via sphingomyelin pathway in the cerebral cortex membranes of mice exposed to stresses exerting diverse effects upon humoral immune response.

The geroprotector activity of epitalon, a synthetic tetrapeptide Ala-Glu-Asp-Gly, was studied on the Drosophila melanogaster wild strain Canton-S. The substance was added to the culture medium only at the developmental stage (from egg to larva). Epitalon significantly increased the lifespan (LS) of imagoes by 11-16% when applied at unprecedented low concentrations-from 0.001 x 10(-6) to 5 x 10(-6) wt.% of culture medium for males and from 0.01 x 10(-6) to 0.1 x 10(-6) wt.% of culture medium for females. The increase in LS did not depend on the substance dose. Effective concentrations of epitalon were 16,000-80,000,000 times lower than those of melatonin. The possible mechanisms of the antioxidant and regulatory effects of epitalon are discussed.