N-Acetyl Epitalon Amidate

Overview of Epitalon-Highly Bioactive Pineal Tetrapeptide with Promising Properties.

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.

Therapeutic Peptides in Orthopaedics: Applications, Challenges, and Future Directions.

Therapeutic peptides are emerging as promising adjuncts in the management of orthopaedic injuries, grounded in their ability to modulate molecular signaling networks central to cellular medicine. By acting on key pathways such as PI3K/Akt, mTOR, MAPK, TGF-β, and AMPK, peptides exert influence over tissue regeneration, inflammation resolution, and neuromuscular recovery. Wound-healing peptides such as BPC-157, TB-500, and GHK-Cu promote angiogenesis, integrin-mediated extracellular matrix remodeling, and fibroblast activation, whereas growth hormone secretagogues like ipamorelin, CJC-1295, tesamorelin, sermorelin, and AOD-9604 activate IGF-1 signaling and satellite cell repair. Recovery-enhancing agents such as epithalon, delta sleep-inducing peptide, and pinealon target circadian and mitochondrial regulators, and neuroactive peptides like selank, semax, and dihexa enhance brain-derived neurotrophic factor and HGF/c-Met pathways critical to neuroplasticity. Although preclinical studies are promising, there is a current lack of clinical trials. This review integrates current mechanistic insights with orthopaedic relevance, emphasizing safety, efficacy, and future directions for responsible integration into musculoskeletal care.

Peptides and Ageing.

A technology has been developed for manufacturing of biologically active complex peptide preparations from extracts of different tissues. In particular, the pineal preparation (Epithalamin) augments the in vitro outgrowth of explants from the pineal gland but not from other tissues, the latter being stimulated by peptide preparations from respective tissues. Epithalamin increases melatonin production by the pineal gland of rats, improves immunological parameters in rats and mice, produces anticarcinogenic effects in different experimental models, stimulates antioxidant defenses, and restores the reproductive function in old rats. These effects are combined in the ability of Epithalamin to increase the lifespan in rats, mice, and fruit flies. Many of these effects are reproduced in clinical trials, which have demonstrated the geroprotector activity of Epithalamin in humans. Among the effects of the thymic preparation Thymalin, those related to its ability to stimulate immunity are the most prominent. This ability is associated with anticarcinogenic and geroprotector activities. Clinical trials of the peptide preparations obtained from other organs including the prostate, the cerebral cortex, and the eye retina, have demonstrated beneficial effects reflected by the improvement of the conditions of respective organs. Based on the data about the amino acid compositions of the peptide preparations, novel principles of the design of biologically active short peptides possessing tissue-specific activities has been developed. Dipeptides specific for the thymus and tetrapeptides specific for the heart, liver, brain cortex, and pineal glands stimulate the in vitro outgrowth of explants of respective organs. Interestingly, for eye retina and the pineal gland, a common tetrapeptide Ala-Glu-Asp-Gly (Epitalon) has been designed, probably reflecting the common embryonal origin of these two organs. Epitalon reproduces the effects of Epithalamin including those related to its geroprotector activity. In particular, Epitalon increases the lifespan of mice and fruit flies and restores the circadian rhythms of melatonin and cortisol production in old rhesus monkeys. At the same time, Epitalon prolongs the functional integrity of the eye retina in Campbell rats with hereditary Retinitis Pigmentosa and improves the visual functions in patients with pigmental retinal degeneration. Changes in gene expression were observed to be produced by the short peptide preparations. Therefore, the effects of Epitalon are suggested to be mediated by transcriptional machinery common for the pineal gland and the retina and, probably, for regulation of melatonin production in fruit flies. Based on three decades of studies of the peptide preparations, the peptide theory of ageing has been put forward. According this theory, ageing is an evolutionary determined biological process of changes in gene expression resulting in impaired synthesis of regulatory and tissue-specific peptides in organs and tissues, which provokes their structural and functional changes and the development of diseases. Correspondingly, correction of such disorders by means of stimulation of peptide production in the organism or through their delivery can promote the normalisation of disturbed body functions.

AEDG Peptide (Epitalon) Stimulates Gene Expression and Protein Synthesis during Neurogenesis: Possible Epigenetic Mechanism.

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.

Epitalon increases telomere length in human cell lines through telomerase upregulation or ALT activity.

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.

The Antioxidant Tetrapeptide Epitalon Enhances Delayed Wound Healing in an in Vitro Model of Diabetic Retinopathy.

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.

Short Peptides Protect Oral Stem Cells from Ageing.

Primary stem cells, after several cell divisions, enter into a senescence state, that is characterized by alterations to spindle-shape typical morphology. This concern is one of the main problems in the use of human mesenchymal stem cells (hMSCs) in clinical applications which demand cells in large numbers. Short peptides had geroprotective properties and stimulated stem cell differentiation. The aim of the study is to demonstrate the role of AEDG and KED peptides in maintaining oral hMSCs morphology and functions over long-term expansion. 2 types of hMSCs were investigated: human periodontal ligament stem cells (hPLSCs) and human gingival mesenchymal stem cells (hGMSCs). Cells at the 25th passage were divided into 3 groups: 1 - control (without adding peptide), 2 - treated with AEDG peptide, 3 - treated with KED peptide. Cell cultures were analyzed by an immunofluorescence method and RT-PCR on the p16 and p21 senescence markers expression. AEDG peptide decreased p16 and p21 mRNA expression by 1.56-2.44 times in comparison with the control group. KED peptide decreased p16 and p21 mRNA expression by 1.82-3.23 times in comparison with the control group. These results were confirmed by immunofluorescent visualization. AEDG and KED peptides could be used as supplementary substances in a culture medium to delay the expression of senescence markers in long term stem cell cultivation in order to promote the large-scale in vitro expansion necessarily required for stem cell therapy clinical application. The data obtained confirm the geroprotective effect of AEDG and KED peptide, which was shown early in animal and cells models.

[AEDG peptide regulates human circadian rhythms genes expression during pineal gland accelerated aging.].

Night work provides biorhythms desynchronization, disorder of melatonin-producing function and accelerated pineal gland aging. One of the promising geroprotectors restoring the pineal melatonin synthesis is the AEDG (Ala-Glu-Asp-Gly) peptide. AEDG peptide increases in 1,7 times the 6-sulfatoxymelatonin (6-SOMT) excretion in the urine of middle-aged people. Moreover, AEDG peptide normalized circadian Clock and Csnk1e genes hyper expression in leukocytes in 1,9-2,1 times and increases the Cry2 gene hypo expression in peripheral blood lymphocytes in 2 times in people with reduced melatonin-producing epiphysis function. The geroprotective effect of the AEDG peptide is based on its ability to restore the epiphysis melatonin-producing function by means regulation of human circadian genes expression.

Epitalon protects against post-ovulatory aging-related damage of mouse oocytes in vitro.

The developmental potential of oocytes decreases with time after ovulation in vivo or in vitro. Epitalon is a synthetic short peptide made of four amino acids (alanine, glutamic acid, aspartic acid, and glycine), based on a natural peptide called epithalamion extracted from the pineal gland. It is a potent antioxidant, comparable to melatonin, that may confer longevity benefits. The current study aims to test the protective effects of Epitalon on the quality of post-ovulatory aging oocytes. Epitalon at 0.1mM was added to the culture medium, and the quality of oocytes was evaluated at 6h, 12h, and 24h of culture. We found that 0.1mM Epitalon reduced intracellular reactive oxygen species. Epitalon treatment significantly decreased frequency of spindle defects and abnormal distribution of cortical granules during aging for 12h and 24h, while increased mitochondrial membrane potential and DNA copy number of mitochondria, thus decreasing apoptosis of oocytes by 24h of in vitro aging. Our results suggest that Epitalon can delay the aging process of oocytes in vitro via modulating mitochondrial activity and ROS levels.

[Aging of the pineal gland].

The age-related changes in the pineal gland are functional rather than organic, which makes their correction or prevention more tenable. The amelioration or inhibition of some age-related impairments of the pineal gland were observed with dietary restriction and the use of S-adenosylmethionine or MAO-A inhibitors. A threefold increase in nocturnal melatonin peaks occurs in old rhesus monkeys treated with a synthetic peptide Ala-Glu-Asp-Gly (Epithalon) designed basing on the amino acid content of a pineal peptide extract Epithalamin. Other effects of Epithalon markedly overlap with melatonin effects. Besides life extension in mice and fruit flies, Epithalon effects include the postponing vision loss in Campbell rats with hereditary pigmental dystrophy. A uniting aspect of such a range of activities might be the participation of transcription factors, since they are often highly conservative in evolution and, on the other hand, may be strictly tissue-specific. The targets of Epithalon may include transfactors that in mammals are specific for the pineal gland and retina and exhibit impaired functions in the aged pineal gland.

Effects of pineal peptide preparation Epithalamin on free-radical processes in humans and animals.

The review on our own data on the effect of the pineal peptide preparation Epithalamin on free radical processes in rodents and humans is presented in this paper. The activity of Cu, Zn-superoxide dismutase (SOD) was found decreased in the brain of aged rats (30 months old) by 46.8% as compared to young animals. Concentration of Schiff's bases in the brain also went down with age (by 13.6%), while the level of dien conjugates (DC) and protein peroxidation (PPO) remained unchanged. General antioxidation activity (AOA) in the brain also remained stable with age. The liver of aged rats showed significant increase of Schiff's bases (by 27.1%) and PPO products (by 109.2%) and considerable decrease of SOD activity. The level of DC and general AOA in the liver remained unchanged with age. Considerable elevation of protein and lipid peroxidation products contents was registered in the blood serum of aged rats. At the same time, general AOA and SOD activity remarkably decreased. The results obtained evidence from both significant age-related alterations in the activity of free radical processes in animal organism and organic peculiarities of their dynamics. Application of peptide drug epithalamin suppressed significantly the intensity of peroxide chemoluminescence in the blood serum (2.8-fold) and lipid peroxide oxidation (LPO) expressed in the considerably decreased DC contents (4,1-fold). The contents of Schiff's bases showed only a tendency towards decrease (by 14.4%, p > 0.05) and PPO level remained unchanged. Epithalamin administration was followed by considerable (by 36.6%, p < 0.01) increase of general AOA and increased SOD activity (by 19.7%) in males. Epithalamin decreased significantly the contents of conjugated hydroperoxides and ketodienes in tissues of D.melanogaster females, increased catalase activity in drosophila males and females, and increased SOD activity in males of D.melanogaster by 41%. Humans reveal significant age-related decrease of antioxidation defence indices. Epithalamin administration to patients with age-related pathology eliminates imbalance in prooxidation and antioxidation systems.

Therapeutic peptides in gerontology: mechanisms and applications for healthy aging.

Peptide therapeutics represent an emerging frontier in gerontological medicine, targeting fundamental hallmarks of aging including metabolic dysfunction, telomere attrition, tissue repair impairment, and hormonal decline. To comprehensively review the mechanisms, clinical applications, evidence base, and safety profiles of therapeutic peptides with demonstrated or potential applications in healthy aging and age-related conditions. A comprehensive narrative review was conducted through systematic searches of PubMed, Scopus, and regulatory databases (FDA, WADA) from inception through January 2026. Search terms included "peptide therapeutics," "aging," "gerontology," "healthspan," combined with specific peptide names (tirzepatide, epitalon, GHK-Cu, BPC-157, TB-500, Semax, CJC-1295, ipamorelin, bremelanotide). Peer-reviewed articles, clinical trials, regulatory documents, and preclinical studies were evaluated. A total of 20 primary sources were selected based on relevance, methodological quality, and contribution to understanding peptide mechanisms and clinical outcomes in aging populations. Nine peptides were identified spanning diverse aging interventions: metabolic restoration (tirzepatide), telomere biology (epitalon), dermal regeneration (GHK-Cu), tissue repair (BPC-157, TB-500), neuroprotection (Semax), growth hormone modulation (CJC-1295, ipamorelin), and sexual function (bremelanotide). FDA-approved agents demonstrated robust safety profiles from large-scale trials. Non-approved peptides showed promising preclinical and limited clinical evidence but lack long-term safety data and systematic validation. Significant knowledge gaps include optimal dosing regimens, combination therapy effects, and biomarkers for monitoring efficacy. Therapeutic peptides offer mechanistically diverse approaches to multiple aging hallmarks. While FDA-approved agents demonstrate clinical potential, investigational peptides require rigorous validation through well-designed clinical trials to establish safety and efficacy for healthspan extension.

Effect of the synthetic pineal peptide epitalon on spontaneous carcinogenesis in female C3H/He mice.

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.

Inhibitory effect of the peptide epitalon on the development of spontaneous mammary tumors in HER-2/neu transgenic mice.

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.

Studies of the effects of Vilon and Epithalon on gene expression in mouse heart using DNA-microarray technology.

Expression of 15,247 clones from a cDNA library in the heart of mice receiving Vilon and Epithalon was studied by DNA-microarray technology. We revealed 300 clones (1.94% of the total count), whose expression changed more than by 2 times. Vilon changed expression of 36 clones, while Epithalon modulated expression of 98 clones. Combined treatment with Vilon and Epithalon changed expression of 144 clones. Vilon alone or in combination with Epithalon activated expression of 157 clones (maximally by 6.13 times) and inhibited expression of 23 clones (maximally by 2.79 times). Epithalon alone or in combination with Vilon activated expression of 194 clones (maximally by 6.61 times) and inhibited expression of 48 clones (maximally by 2.71 times). Our results demonstrate the specific effects of Epithalon and Vilon on gene expression.

Pineal peptide preparation epithalamin increases the lifespan of fruit flies, mice and rats.

Treatment with pineal peptide preparation epithalamin was followed by the increase of the mean lifespan of female D. melanogaster, SHR mice, C3H/Sn mice and LIO rats by 11-31% (P < 0.05). Ninety percent mortality as well as maximum lifespan were increased in fruit flies, C3H/Sn mice and rats. Mortality rate was decreased by 52% in D. melanogaster, by 52% in rats, by 27% in C3H/Sn mice. It did not change in SHR mice exposed to epithalamin. Treatment with the pineal peptide increased MRDT in flies, C3H/Sn mice and rats. It has been shown that epithalamin increased synthesis and secretion of melatonin in rats and inhibits free radical processes in rats and in D. melanogaster. It is suggested that antioxidative properties of epithalamin lead to increased lifespan of three different animal species.

Inhibitory effect of peptide Epitalon on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats.

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.

[Geroprotective effect of thymalin and epithalamin].

Researchers of the St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the Russian Academy of Medical Sciences and the Research Institute of Gerontology of the Ukrainian Academy of Medical Sciences (Kiev) clinically assessed the geroprotective effects of thymic and epiphyseal peptide bioregulators (Thymalin and Epithalamin, correspondingly) in 266 elderly and older persons during 6-8 years (the bioregulators were applied for the first 2-3 years of observation). The obtained results convincingly confirmed the ability of the bioregulators to normalize the basic functions of the human organism, i.e. to improve the indices of the cardiovascular, endocrine, immune, and nervous systems, homeostasis, and metabolism. The restoration of homeostasis in the patients was accompanied by a 2.0-2.4-fold decrease in acute respiratory disease incidence, reduced incidence ischemic heart disease clinical manifestations, hypertension, deforming osteoarthrosis, and osteoporosis, as compared to the control group. Such a significant improvement in the somatic state of the peptide-treated patients corresponded to a decrease in their mortality rate during the observation period: 2.0-2.1-fold among the Thymalin-treated patients, 1.6-1.8-fold--in the Epithalamin-treated group, and 2.5-fold--in the patients treated with Thymalin combined with Epithalamin, as compared to the control group. A separate group of patients was treated with Thymalin combined with Epithalamin annually for 6 years. We registered a 4.1-fold mortality decrease in this group as compared to the control level. The results of our research confirmed the conclusion on the high geroprotective efficacy of Thymalin and Epithalamin and the expediency of their application in medicine and social care as the means of health maintenance and age-related pathology prevention in persons over 60 years old enabling the prolongation of the active period of their lives.

Modulating effects of epithalamin and epithalon on the functional morphology of the spleen in old pinealectomized rats.

Immunohistochemical and morphometric analysis showed that epithalamin and epithalon produced similar effects on the functional morphology of the spleen in pinealectomized rats. Both peptides prevented hyperplasia of lymphoid cells in follicular germinative centers induced by pinealectomy and potentiated the decrease in extramedullary hemopoiesis. These findings confirm the data on functional relationships between the pineal gland and immune system. The effects of epithalamin and epithalon on cell and tissue homeostasis in the spleen of old pinealectomized rats can be regarded as a manifestation of the general regulatory effect of these peptides.

Effect of melatonin and pineal peptide preparation epithalamin on life span and free radical oxidation in Drosophila melanogaster.

It was shown previously that epithalamin delays age-related changes in reproductive and immune systems and increases the life span of mice and rats. These effects could be mediated by stimulating influences of epithalamin on synthesis and secretion of melatonin and on free radical processes. A comparative study on the effect of epithalamin and melatonin on both the life span of Drosophila melanogaster (strain HEM) and on the intensity of lipid peroxidation and activity of antioxidative enzymes in their tissues was the main aim of this work. Melatonin and epithalamin was added to the nutrition medium (100 micrograms/ml) during 2-3rd age of larvas. For survival analysis the flies were passed (five coupes per vessel) each 3-7 days. Lipid peroxidation was evaluated as the level of ketodienes (KD) and conjugated hydroperoxides (CHP) in fly tissues at the age of 11 days. Activity of Cu, Zn-superoxide dismuatse (SOD) and catalase was evaluated as well. The mean, median and maximum life span (MLS) were estimated. Mortality rate (MR) was calculated as alpha in the Gompertz equation (R = Ro (exp alpha t) and mortality rate doubling time (MRDT) as in 2/alpha. These parameters in groups of male and female flies exposed to melatonin and in male flies exposed to epithalamin were no different from the parameters for controls. However, exposure to epithalamin was followed in females by a significant increase in mean life span (by 17%, P < 0.02), of median (by 26%), of MLS by 14% and by a 2.12 times decrease of MR (P < 0.01) and MRDT (by 32%) compared with female controls. The level of CHP and KD in the tissues of male control flies was 40 and 49% less than that in females and indirectly correlates with male life span. Exposure to melatonin was followed by a decrease in the level of CHP and KD in females and the deletion of sex differences in them. Exposure to epithalamin significantly decreased the level of CHP and KD in female flies compared to controls (2.3 and 3.4 times, respectively, P < 0.001). Exposure to melatonin failed to influence the activity of catalase in males but increased it in females by 24% (P < 0.02) and failed to influence SOD activity both in males and females. Exposure to epithalamin was followed by a significant increase in activity of catalse, 20% in males and 7% in females and by an increase in SOD activity in males (41%). Thus, it was shown that exposure to epithalamin significantly increases the mean life span and MLS of female D.melanogaster and slowed down their aging rate by 2.12 times. This effect is in good agreement with the inhibiting effect of epithalamin in lipid peroxidation processes in fly tissues.

Epithalon decelerates aging and suppresses development of breast adenocarcinomas in transgenic her-2/neu mice.

Female transgenic FVB/N mice carrying the breast cancer gene HER-2/neu received epithalon (Ala-Glu-Asp-Gly) in a dose of 1 mg subcutaneously 5 times a week to from the 2nd month of life to death. Epithalon prolonged the average and maximum lifetimes of mice by 13.5 (p<0.05) and 13.9%, respectively. The peptide prolonged the average lifetime of animals without neoplasms (by 34.2%, p<0.05). Epithalon decelerated the development of age-related disturbances in reproductive activity and suppressed the formation of neoplasms. The peptide decreased the incidence of breast adenocarcinomas, lungs metastases (by 1.6 times, p<0.05), and multiple tumors (by 2 times). Epithalon 3.7-fold increased the number of mice without breast tumors (p<0.05), while the number of animals with 6 or more breast tumors decreased by 3 times (p<0.05). Epithalon prolonged the lifetime of mice with breast tumors by 1.4 times (p<0.05). These results indicate that Epithalon possesses geroprotective activity and inhibits breast carcinogenesis in transgenic mice, which is probably related to suppression of HER-2/neu expression.

Effects of intranasal administration of epitalon on neuron activity in the rat neocortex.

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.

Twenty years of study on effects of pineal peptide preparation: epithalamin in experimental gerontology and oncology.

Artificial life extension. The epigenetic approach.

An epigenetic approach starts out from the direct (rather than the underlying genetic) causes. An epigenetic approach to aging has little chance of succeeding before a minimum amount of knowledge has been accumulated on the "genetic programming" that is currently believed to underlie aging. Two recent advances, one empirical and one theoretical, jointly brighten the prospect. The empirical one is the discovery that melatonin functions as an aging-controlling hormone in mammals. In 1979, Dilman and co-workers isolated a biologically active pineal extract (epithalamin) in rats which, as they later showed, stimulates melatonin production. Pierpaoli and co-workers in 1987 directly administered melatonin to mice. Both groups observed a surprising 25-percent increase of life span in conjunction with a postponed senescence. A similar effect was also achieved with an engraftment of young pineal tissue into the thymus of old mice by Pierpaoli's group. Beneficial effects of epithalamin in humans were reported by Dilman's group. The second advance is a deductive evolution-theoretical approach to aging discovered in 1988. In populations living in a niche with a fixed carrying capacity, any individual is in the long run replaced by a single successor. It follows that, as the expected cumulative number of adult progeny of the same sex approaches unity as a function of life time of the progenitor, the latter's survivability must approach zero if the sum is to remain unity. A physiological prediction follows: a centralized physicochemical clock--like a sedimentation process--must exist somewhere in the organism controlling a secreted substance that reaches all cells. In this way, the pineal coacervates and the pineal's hormonal product melatonin were arrived at on an independent route again. While melatonin as a drug has been used on human volunteers for decades, its anti-aging effect has yet to be proved. Detailed hormone profiles in different age groups and under different life styles have to be performed. A modified Hayflick in vitro experiment is also needed to elucidate the mechanism by which melatonin works in cells.

Epitalon and colon carcinogenesis in rats: proliferative activity and apoptosis in colon tumors and mucosa.

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.

Effect of Ala-Glu-Asp-Gly peptide on life span and development of spontaneous tumors in female rats exposed to different illumination regimes.

The effects of Ala-Glu-Asp-Gly peptide (Epithalon) on the life span and development of spontaneous tumors were studied in female rats exposed to standard, natural for North-Western Russia, and constant illumination. The mean life span of animals exposed to constant or natural illumination decreased by 13.5 and 25.5%, the maximum by 9 and 7 months, respectively, and spontaneous tumors developed much more rapidly than in animals living under conditions of the standard light regimen. Epithalon (0.1 microg daily 5 times a week from the age of 4 months) did not change the life span of rats living under conditions of standard day/night regimen, while in rats exposed to the natural and constant light it promoted prolongation of the maximum life span by 95 and 24 days, respectively. Epithalon prolonged the mean life span of the last 10% of rats exposed to natural and constant illumination, treated with Epithalon, by 137 and 43 days, respectively. This peptide exhibited virtually no effect on the development of spontaneous tumors in rats exposed to standard and constant illumination, but significantly inhibited their development in rats exposed to natural light.

Epitalon-activated telomerase enhance bovine oocyte maturation rate and post-thawed embryo development.

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&#xa0;<&#xa0;0.05). Furthermore, telomerase activity was significantly compromised in post-thawed embryos, and Epitalon treatment significantly improved blastocyst hatching rate and implantation potential (p&#xa0;<&#xa0;0.05). Moreover, we performed qPCR, reactive oxygen species, and JC-1 (&#x394;&#x3a8;m) 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.

Epitalon influences pineal secretion in stress-exposed rats in the daytime.

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. Intact and osmotic-stress-exposed rats were used for the immunohistochemical detection of C-Fos protein. All animals were intranasally administered with Epitalon, the last infusion made in two hours before the biopsy. Simultaneously, light microscopy of the pineal parenchyma was performed in all groups of animals. A slight but significant C-Fos increase was observed only in stress-exposed pinealocytes of rats after intranasal Epitalon infusions. C-Fos was irregularly distributed throughout pineal cells. In stress, the clusters of 5 10 cells containing C-Fos in their cytoplasm were detected. The dilation of capillaries and pericapillary space induced by an osmotic stress was partially reduced by the intranasal infusions of Epitalon. Tetrapeptide Epitalon is synthesised on the basis of the amino acid composition of pineal peptide extract Epithalamin. Epitalon modulates pineal secretion only under a stress impact but never in the norm. It prevents osmotic-stress-induced pathologic changes in the pineal parenchyma structure. Besides, the physiological activity of Epitalon seems to be mediated by the activation of protooncogenes in pinealocytes.

[Effect of epitalon and melatonin on life span and spontaneous carcinogenesis in senescence accelerated mice (SAM)].

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.

Peptide Epitalon activates chromatin at the old age.

OBJECTIVES and design. We have studied the effect of synthetic peptide Epitalon on the activity of ribosomal genes, denaturation parameters of total heterochromatin, polymorphism of structural C-heterochromatin and the variability of facultative heterochromatin in cultured lymphocytes of persons aged 76-80 years. The obtained data demonstrate that Epitalon induces the activation of ribosomal genes, decondensation of pericentromeric structural heterochromatin and the release of genes repressed due to the age-related condensation of euchromatic chromosome regions. Epitalon has shown its ability to activate chromatin by modifying heterochromatin and heterochromatinized chromosome regions in the cells of older persons.

Tissue-specific effects of peptides.

Synthetic peptides (cytogens) Cortagen, Epithalon, Livagen, and Vilon stimulated the growth of explants from rat brain cortex, subcortical structures, liver, and thymus, respectively, in organotypic cultures. These peptides produced tissue-specific effects: they stimulated the growth of explants from tissues, whose cytomedins (peptide complexes) were used for chemical synthesis.

Epithalon peptide induces telomerase activity and telomere elongation in human somatic cells.

Addition of Epithalon peptide in telomerase-negative human fetal fibroblast culture induced expression of the catalytical subunit, enzymatic activity of telomerase, and telomere elongation, which can be due to reactivation of telomerase gene in somatic cells and indicates the possibility of prolonging life span of a cell population and of the whole organism.

[Intranasal epitalon infusion modulates neuronal activity in the rat neocortex].

Properties of tetrapeptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of pineal peptide extract, have been studied. The intranasal infusions: a noninvasive way to deliver this peptide to CNS hypassing the blood-brain barrier, was used. The aim of the study is to estimate epitalon action on rat motor cortex spontaneous activity. Wistar male rats were anesthetized with urethane (1 g/kg). Extracellular unit recording was made using glass microelectrodes (1-2 MOhm). After recording of spontaneous activity (10-15 min), epitalon intranasal infusion (2 ng) was followed by 30-minute recording. Within a few minutes after the infusion, significant activation of neural activity was observed (2-2.5-fold higher frequency of neuronal spikes). Complex response consisting of several phases was identified in some recordings. The spikes frequency growth during 5 to 7 min (first phase) after the infusion was followed by the second (11-12 min) and the third (17-18 min) phases. An increase of neuronal spontaneous activity was conditioned by the higher frequency of already active units and by the involvement of previously silent cells. At least the first phase of epitalon action can be explained by direct action of the peptide on the cells of the motor cortex.

Effect of Epitalon on biomarkers of aging, life span and spontaneous tumor incidence in female Swiss-derived SHR mice.

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.

Effect of peptide preparation epithalamin on circadian rhythm of epiphyseal melatonin-producing function in elderly people.

Circadian rhythm of plasma melatonin concentrations in healthy elderly subjects was studied before and after a course treatment with Epithalamin (peptide preparation from the pineal gland). Epithalamin modulated the melatonin-producing function of the pineal gland. During the dark period plasma melatonin concentration increased in subjects with initially lowered activity of the pineal gland, while in subjects with normal epiphyseal function plasma melatonin concentration tended to decrease.

Effect of epitalon on the lifespan increase in Drosophila melanogaster.

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.

Anti-aging peptide bioregulators induce reactivation of chromatin.

The effect of synthetic peptide bioregulators (Epitalon, Livagen and Vilon) on structural and facultative heterochromatin of cultivated lymphocytes have been studied among old (75-88yr.) people. The data obtained indicate that epitalon, livagen and vilon: 1) activate synthetic processes, caused by reactivation of ribosomal genes as a result of deheterochromatinization (decondensation) of nucleolus organizer regions; 2) induce unrolling (deheterochromatinization) of total heterochromatin; 3) release genes repressed by heterochromatinization (condensation) of euchromatic regions forming facultative heterochromatin; 4) epitalon and livagen induce deheterochromatinization (decondensation) of pericentromeric structural heterochromatin of the chromosomes1 and 9. However, vilon does not induce deheterochromatinization of pericentromeric structural heterochromatin. These results indicate that peptide bioregulators Epitalon, Livagen and Vilon cause activation (deheterochromatinization) of chromatin in lymphocytes of old individuals.

Effect of epithalon on age-specific changes in the retina in rats with hereditary pigmentary dystrophy.

The effect of peptide bioregulator Epithalon on the course of hereditary pigmentary retinal degeneration was studied in Campbell rats. Administration of epithalon starting from birth protected morphological structure, increased its bioelectrical activity, and improved its function.

Effect of melatonin and tetrapeptide on gene expression in mouse brain.

A microchip technique was used to study expression of 16,897 clones from a cDNA library in the brain of mice receiving melatonin or tetrapeptide Epithalon (Ala-Glu-Asp-Gly). Expression of 53 transcripts in mouse brain underwent significant changes after treatment with the preparations. Melatonin and Epithalon modified expression of 38 and 22 transcripts, respectively. These preparations produced similar changes in the expression of 6 transcripts. Expression of 1 transcript (Rp119) was inhibited by melatonin, but induced by Epithalon. The target genes are physiologically related to the cell cycle, apoptosis, biosynthesis, processing, and transport of nucleic acids. Comparative study of gene expression in the brain and heart of CBA mice receiving melatonin and Epithalon suggest that these preparations have a tissue-specific biological effect.

Peptides of pineal gland and thymus prolong human life.

Researchers of the St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the Russian Academy of Medical Sciences and the Institute of Gerontology of the Ukrainian Academy of Medical Sciences (Kiev) clinically assessed the geroprotective effects of thymic (Thymalin) and pineal (Epithalamin) peptide bioregulators in 266 elderly and older persons during 6-8 years. The bioregulators were applied for the first 2-3 years of observation. The obtained results convincingly showed the ability of the bioregulators to normalize the basic functions of the human organism, i.e. to improve the indices of cardiovascular, endocrine, immune and nervous systems, homeostasis and metabolism. Homeostasis restoration was accompanied by a 2.0-2.4-fold decrease in acute respiratory disease incidence, reduced incidence of the clinical manifestations of ischemic heart disease, hypertension disease, deforming osteoarthrosis and osteoporosis as compared to the control. Such a significant improvement in the health state of the peptide-treated patients correlated with decreased mortality rate during observation: 2.0-2.1-fold in the Thymalin-treated group; 1.6-1.8-fold in the Epithalamin-treated group; 2.5-fold in the patients treated with Thymalin plus Epithalamin as compared to the control. A separate group of patients was treated with Thymalin in combination with Epithalamin annually for 6 years and their mortality rate decreased 4.1 times as compared to the control. The obtained data confirmed the high geroprotective efficacy of Thymalin and Epithalamin and the expediency of their application in medicine and social care for health maintenance and age-related pathology prevention in persons over 60 to prolong their active longevity.