Overview of Epitalon—Highly Bioactive Pineal Tetrapeptide with Promising Properties

Over 50 years ago, in 1973, the term Epithalamin was used for the first time in widely accessible scientific research. It describes a pineal polypeptide extract from cattle, which has been found to have unique properties in in vitro and in vivo experiments [ 1 ]. The discovery of the bioactivity of this extract and its first research were mainly led by V.K Khavinson and V.N Anisimov [ 2 ]. Since then, Epithalamin has been widely studied, including in clinical trials, showing a normalizing effect on the basic functions of the human organism and, as a result, establishing it as a geroprotective agent [ 3 ]. Despite the publication of several reviews of Epithalamin [ 4 , 5 , 6 , 7 ], to the best of our knowledge, none specifically concentrate on Epitalon. Epitalon, also known as Epithalon or Epithalone, is a tetrapeptide that was developed on the basis of the amino acids composition of Epithalamin [ 8 ]. For many years, the presence of Epitalon in the human body remained unconfirmed, until 2017, when it was detected for the first time in physiological pineal gland extract [ 9 ]. This explains why Epitalon has similar properties to Epithalamin, but differs in the intensity of some actions [ 10 , 11 ]. It should be noted that some authors do not use this common name of Epitalon and refer only to its primary structure—AEDG. This name highlights the fact that Epitalon is a tetrapeptide consisting of Ala-Glu-Asp-Gly (AEDG) amino acids bound through α-peptide bonds ( Figure 1 a). Interestingly, in one article, where the antioxidant properties of Epitalon were investigated, the authors presented a different chemical structure of Epitalon, composed of the same amino acids but bonded in a different way [ 12 ]. In this work, the peptide bond between glutamic acid and aspartic acid was through the δ-carboxylic group and the peptide bond between aspartic acid and glycine was made by the γ-carboxylic group ( Figure 1 b). The authors named this variant of Epitalon Ala—γ Glu—γ Asp—Gly, which indicates a potential naming mistake. However, this structure of “double-gamma bonded” Epitalon is rather an exception, as we have not encountered it in any other of the published works. Since Epitalon has a natural origin, it is mostly investigated in its free-base form. However, some authors have specified that Epitalon in the form of salt was used in their experiments. Most common salts of Epitalon available commercially are in combination with acetic or trifluoroacetic (TFA) counterions. Epitalon, owing to its significant bioactivity and peptide composition, has primarily been studied in vivo through subcutaneous injections. However, some sources assert that Epitalon is a low-hydrolysable peptide that can be taken orally, exhibiting unique features [ 13 ]. This study intends to perform a critical evaluation of the published data on Epitalon, encompassing all experimental types—in silico, in vitro, and in vivo. The precise mechanism of action of this tetrapeptide remains unverified, prompting a diversification of the research on both the methodologies employed and the criteria examined. Consequently, we deemed it essential to consolidate the acquired knowledge on Epitalon, not merely as a summary, but also to facilitate the planning of future studies on this interesting peptide.

D. melanogaster has been identified as a valuable model for assessing lipid peroxidation levels. Highly inbred strains were selected by the reduced mating potential of males. Epitalon was added into the nutritional medium at the larval stage at a concentration of 0.00001% ( w / w ). The results indicated that adult flies had reduced amounts of conjugated hydroperoxides following Epitalon treatment during the larval stage. An identical outcome was also seen for the concentrations of Schiff’s bases. The results varied by gender, although in both instances, the observed alterations following Epitalon administration indicated reduced concentrations. These results demonstrate that Epitalon undoubtedly possesses antioxidant properties. Furthermore, the authors indicated that, upon comparison with the literature, the effective concentration was sufficiently low to suggest a signaling role for Epitalon in the cascades of the cellular antioxidant system [ 57 ]. The second article by the same authors from the same year additionally investigated catalase activity. Again, Epitalon had antioxidating effects. Additionally, Epitalon was compared with its natural occurring progenitor, Epithalamin. The results of the comparative analysis in this study revealed that Epitalon is a better antioxidant than Epithalamin. It is important to note that Epithalamin was administered at 1000-fold higher doses [ 58 ]. A similar experiment focused on measures of reactive oxygen levels in flies. Epitalon was compared with Vilon. Both peptides inhibited reactive oxygen formation in mitochondria and cytosol [ 59 ]. Epitalon has an influence on the lifespan of D. Melanogaster. A wild strain, Cantos-S., of D. Melanogaster was used as a model in an experiment, where early developmental stages were treated with Epitalon at different concentrations. Overall, Epitalon increased the lifespan of imago D. Melanogaster by up to 16%. This effect was observed for both genders. Survival curves additionally confirmed that Epitalon mainly influenced mature and old imago flies. Khavinson et al. stated that these results confirmed the lack of genotoxic effects potentially conducted by Epitalon [ 60 ]. What was special about Epitalon were the doses which induced the effect. To achieve comparable results for a medium life span extension for D. Melanogaster, melatonin has to be implemented at 16,000-fold higher concentrations [ 61 ].

The majority of articles mentioning Epitalon use rats as model animals for in vivo experiments. Similarly to experiments performed on mice, many strains were used and widely investigated. Epitalon was found to be a gastric endocrine cell behavior-modifying agent [ 77 , 78 ]. In a study performed on Wistar rats, before Epitalon subcutaneous injection, rats were pinealectomized. Epitalon was administered for 30 or 42 days. Serotonin-producing enterochromaffin cells, gastrin-producing cells, and somatostatin-producing cells were counted. The number of above-mentioned cells in pinealectomized rats without Epitalon administration did vary significantly compared to controls. Epitalon did, to a certain degree, decrease the effect of pinealectomy on gastric cells. For example, the ratio between gastrin- and somatostatin-producing cells stayed at the same level as those in the control group after 30 days of Epitalon administration. Forty-two days of subcutaneous Epitalon administration fully recovered the pinealectomy-induced changes in gastric endocrine cells to the levels of the control group. This experiment led V.K. Khavinson et al. to the conclusion that Epitalon has a direct impact on gastric endocrine cells [ 78 ]. Another study examined the characteristics of the rat duodenum following γ-irradiation. Intraperitoneally administered Epitalon inhibited proliferation and exhibited no additional post-γ-irradiation activity. The authors stated that such activity of Epitalon could be beneficial in antitumor treatments [ 79 ]. This statement stays in line with the results obtained by V.N. Anisimov et al., where Epitenon significantly decreased the amount of cancer incidence in a 1,2-dimethylhydrazine-induced colon carcinogenesis model [ 80 ]. In 2003, the anti-cancer activity of Epitalon was confirmed in different developmental stages of colon cancer. Epitalon is especially effective when applied before, during, and after cancer occurrence [ 81 ]. Epitalon also exerted a regulatory influence on pepsin activity during aging in unfavorable light conditions of the Karelia Republic. Nevertheless, it was demonstrated that Epitalon did not significantly affect overall proteolytic activity. Research demonstrated that pepsin activity and overall proteolytic activity remained unchanged by Epitalon under 12 h light/dark cycles or constant light [ 82 ]. The impact of pinealectomy on the spleen was similarly examined. Subcutaneous injections of Epitalon or Epithalamin were administered to pinealectomized rats. Similar to the previous experiment involving intestinal cells, Epitalon and Epithalamin only partially mitigated the impact of pinealectomy on spleen cells. It was confirmed that Epitalon and Epithalamin significantly affect splenic functionality and morphology. The results for groups administered with Epitalon and Epithalamin exhibited statistically significant differences compared to the control group and rats without splenic activity adjustment [ 10 ]. As previously mentioned, Epitalon shows geroprotective activity [ 83 ]. This activity is usually connected with antitumor activity. This was also confirmed in an experiment performed on male LIO rats, where tumor incidence depending on Epitalon administration and light exposure was investigated. Epitalon did significantly lower tumor incidence in all three light exposure schemes ( p < 0.02). However, Epitalon did not prolong the mean lifespan in all groups [ 84 ]. Tumor incidence dependence on Epitalon administration and light exposure was also investigated in female LIO rats. In this experiment, rats exposed to light natural for northwestern Russia and constant light showed a prolongation of their maximum life span. The results also presented that spontaneous tumor incidence for female rats was lowered only in natural northwestern Russia light conditions [ 85 ]. Another anti-aging characteristic of Epitalon, previously emphasized, is anti-free-radical oxidation inhibition. Based on the lipid peroxidation rates in the brain and serum of Wistar rats, this property of AEDG was confirmed in vivo in rats by L.S. Kozina [ 1 ]. The next anti-aging related property of Epitalon is the inhibition of apoptosis. To validate this property in rats, Wistar rats were subjected to γ-irradiation, and the apoptosis rate of their splenic lymphocytes was assessed. On day 2 post-irritation, Epitalon was delivered intraperitoneally with physiological saline to one group for the subsequent 5 days. Epitalon reduced the apoptosis of splenic cells by 2.12-fold ( p < 0.05) compared to the control group that received only saline injections [ 86 ]. A separate investigation utilizing γ-irradiation to examine the protective qualities of Epitalon in rats yielded noteworthy results. Microscopic examination revealed that Epitalon partially preserved the pineal gland from structural changes and facilitated its recovery, particularly through the partial restoration of the mitochondrial ultrastructure

Previous promising results on Campbell rats, where Epitalon was found to be a suitable drug for Retinitis pigmentosa [ 18 , 19 , 20 ], led to a clinical trial. The trial was performed at the St. Petersburg Institute of Bioregulation and Gerontology. Overall, 162 patients with Retinitis pigmentosa , aged from 18 to 72 years, were examined. Each patient received 5.0 µg of Epitalon per eye, injected parabulbarly for 10 consecutive days. The control group was treated with conventional methods for 2002 (antisclerotic agents, vasodilators, and angioprotectors). None of the Epitalon-treated patients reported side effects. The amplitude activity of both the first and second neurons rose significantly. The visual acuity of treated patients increased by 0.15–0.20 on average. The peripheral borders of the visual field were extended in all patients. A total of 64.8% of patients had their total visual field border broadened by 90–120 degrees. Absolute scotomas reduced in size and some of them even disappeared. These results evidence that Epitalon treatment is suitable for Retinitis pigmentosa treatment [ 20 ]. A separate study conducted on 75 women examined the ability of Epitalon to regulate and preserve the circadian rhythm. Epitalon was delivered sublingually for a duration of 20 days at a dose of 0.5 mg/day. The production of melatonin in the epiphysis and the expression of human circadian genes were examined. The research comprised placebo and control cohorts. Melatonin synthesis in the epiphysis was assessed by quantifying the excretion of 6-sulfatoxymelatonin in urine. Epitalon was observed to enhance this parameter by 1.6 times relative to the placebo group. The circadian genes examined were the Clock and Cry2 genes in leukocytes and the Csnk1e gene in lymphocytes. Clock gene expression was reduced by a factor of 1.8 relative to the placebo. Following the administration of Epitalon, the Cry2 expression in leukocytes was doubled ( p < 0.05) compared to pre-treatment levels, while the Csnk1e expression in blood cells decreased by 2.1 times ( p < 0.05). The scientists concluded that Epitalon’s geroprotective activity is attributed to its capacity to restore epiphyseal melatonin production through the modulation of human clock gene expression [ 108 ].

Apart from in vitro and in vivo experiments, Epitalon has also been studied using in silico approaches. Epitalon’s molecular properties in aqueous solution were calculated at the molecular mechanics level [ 29 , 113 ]. Molecular dynamics simulations of Epitalon in zwitter-ionic form were conducted using an AMBER force field, with a 37.5 Å × 22.5 Å × 13.5 Å rectangular box containing 347 solvent molecules. A TIP3P model was used to represent water molecules during calculations. The simulation lasted 1500 ps and the temperature of the calculated system was set to 300 K. The conformer structure with the lowest energy and 120 other conformers were obtained. It was found that Epitalon forms two intramolecular salt bridges which stabilize its structure. Both of these salt bridges are formed between the nitrogen atom of alanine and non-peptide-bond-forming carboxylic groups of glutamic acid and aspartic acid. Additionally, an intramolecular hydrogen bond was recognized. Rogachevskii et al. stated that these intramolecular bonds highly decrease the conformational freedom of Epitalon [ 114 ]. Epitalon was also studied in silico in 2003 by investigating its interactions with DNA fragments. Epitalon was found to “unbound” to a β-like structure and interact with a specific ATTTG DNA sequence by hydrophobic interactions and hydrogen bonds. Hydrophobic forces were observed between the side chains of the peptide and the methyl groups of thymine. However, this article provided no precise data about the structure of such a complex [ 113 ]. The topic of DNA binding was further discussed in 2004. Again, Epitalon was found to interact with a specific DNA sequence—this time, ATTTC. A 3D model of this complex was not provided by the authors, but a more specific “interactions graph” was presented ( Figure 6 ). The exact consequence of the binding of this molecule to this particular base sequence is not known, however, Khavinson V.K. et al. correlated this characteristic of Epitalon with its specific biological action, especially because this specific sequence is present multiple times in the promoter part of the telomerase gene [ 115 ]. Investigations into the thermodynamics of DNA have demonstrated that incremental temperature elevations lead to a phase shift in the “helix–coil”, also known as DNA melting [ 116 ]. This phase transition causes the double helix to dissociate into two single strands, manifesting as random coils. A comparable reversible helix–coil transformation transpires at ambient temperature, without thermal application, when DNA is subjected to acidic and alkaline solutions. This random coil transition is associated with an increase in absorbance at 260 nm, referred to as the hyperchromic effect [ 117 ]. A. Solovyev et al. discovered that the presence of Epitalon reduced the melting temperature of double-stranded DNA by as much as 41 °C at an ionic strength equivalent to a 0.1 M NaCl concentration [ 118 ]. The molecular docking of Epitalon to other molecular targets was also performed. In 2013, Fedoreyeva et al., based on an experiment that revealed interactions between Epitalon molecules and labeled histones, proposed that the epigenetic effect of this molecule is not only related to interactions with DNA [ 119 ]. Seven years later, these results led to an Epitalon/histone complex Molecular Dynamics (MD) analysis, based on Molecular Mechanics (MMs), using an Amber12EHT force field. Overall, 50 docking results with 6 different histone proteins were investigated. The core histones H2b, H3, and H4 did not reveal any binding sites suitable for Epitalon. It was found that Epitalon most likely forms complexes with histones H1/6 (binding energy: −64.51 kcal/mol) and H1/3 (binding energy: −56.49 kcal/mol). This is especially important, because these proteins interact with DNA helixes through these sites. This is consistent with the thesis stated before [ 40 ]. A subsequent molecular docking experiment was conducted by docking Epitalon to the transporter proteins LAT1, LAT2, PEPT1, and PEPT2. Values for the scoring functions of Epitalon indicating strong binding, represented by the ICM-Score, were acquired ( Table 4 ). Epitalon has a binding strength comparable to that of established PEPT1 inhibitors. The authors of this work indicated that the anti-cancer capabilities of Epitalon, previously demonstrated in in vitro and in vivo experiments, may be associated with the blockage of the amino acid transporters LAT1, LAT2, and PEPT1 [ 120 ]. The possibility of Epitalon forming complexes with lysine dendrimers was also investigated in silico using MD. The time taken for the evolution of a system containing 16 molecules of Epitalon with second- and third-generation lysine dendrimers in a water environment was investigated. Simulations lasting 160 ns showed the complete absorption of all peptide molecules on the dendrimer. Complexes between 16 Epitalon molecules and second-generation dendrimers were formed after 20 ns. The ra

As shown in this review, Epitalon exhibits significant and multidirectional pharmacological activity. However, in the analyzed works, information regarding critical issues about this peptide’s safety is missing. Before Epitalon’s approval as a new API, additional studies on its potential short- and long-term toxicity are essential [ 56 ]. These future studies should also include aspects such as genotoxic activity, carcinogenic potential, and food–drug and drug–drug interactions. Furthermore, short peptides such as Epitalon are typically unstable and degrade rapidly in vivo. Chemical modifications like acetylation and amidation can sometimes be employed to enhance the stability of such peptides. However, to the best of our knowledge, in the case of Epitalon, this idea has not been evaluated yet. Also, due to the presence of three asymmetric centers, Epitalon exists in the form of eight stereoisomers. While the naturally occurring tetrapeptide consists solely of L-amino acids, a good research direction would be to evaluate the pharmacological properties of the other seven stereoisomers in a comparative study.