Peptide Self-Regulation of Living Systems (Facts and Hypotheses) - Shataeva L. K. 2003

Peptide Regulation of Aging
Theoretical and Experimental Prerequisites for the Correction of Age-Related Pathology

The medical challenges of treating geriatric diseases were first framed in classical antiquity by Hippocrates, further elaborated by Avicenna in The Canon of Medicine, developed in medieval medicine, and are currently being investigated in depth at the modern level of chemistry, pharmacology, and medicine.

It is evident that a healthy old age preserves a substantial reserve of bodily resources to overcome the consequences of disease. However, these resources can be rapidly depleted by psychological stress. In particular, normal physiological Aging proceeds at an individual rate for each person: physical and mental Functions decline gradually over many years. Abruptly uprooting an individual from an active and familiar social routine (for example, As a result of retirement) does not match the pace of the body's slower aging process, leading to significant psychological stress that frequently triggers secondary illnesses (WHO Working Group, 1982). For normal longevity, the gradual shift in an elderly person's social status must correspond to The rate of change in their functional status. In this context, psychological preparation for the inevitability of the end of life is of critical importance. If we are threatened by misfortune (and death is invariably viewed as a misfortune), There are two possible courses of action: one can attempt to avoid the misfortune, or one can face it with dignity. The first approach—overcoming personal death—remains an indefinite prospect. The second approach opens up to every person sooner or later. Normal physiological aging helps to perceive the end of life not merely as a necessity, but as a personal blessing.

The Role of the Brain and consciousness was confirmed by an experimental comparison of the efficacy of drug-based and drug-free assistance programs for the elderly. The drug-free program involved memory training and The stimulation of patients' cognitive abilities to compensate for and overcome age-related memory decline. Instead of active medications, they received a placebo. The control group received only active medications. The effectiveness of these two Methods was evaluated through independent patient examinations and the patients' own subjective Assessment of the results. Objective Examination demonstrated comparable efficacy between the pharmacological and non-pharmacological methods. When asked, "Do you consider this Treatment effective? To what do you attribute your improvement—the pills or the memory training?" the majority of patients replied that it was due to the memory training (Israel et al., 1987).

Apparently, Structure/19.html">The Importance of intellectual workload as a strengthening exercise for the elderly has not yet been properly appreciated in practical gerontology. Moreover, recommendations for improving the well-being of older adults generally boil down to a rational low-calorie diet, lowered body Temperature, and specialized physical exercises (Walford et al., 1980; Korkushko et al., 2002). In other words, weight control and physical activity are considered sufficient means for preventing aging. At the same time, standard nutritional guidelines are calculated based on the caloric content of foodstuffs determined by their Heat of Combustion in a calorimeter under an oxygen atmosphere (Skurikhin, Nechaev, 1991). Obviously, a low-calorie diet determined in this manner is ill-suited for elderly individuals, whose age brings about an altered individual balance of digestive Enzymes, changes in glycocalyx activity, and shifts in intestinal endothelium permeability (Galperin, Lazarev, 1986). Furthermore, modern rational Nutrition schemes fail to account for the body's age-increasing requirements for protein foods balanced in Amino Acid Composition, native Peptides, and Nucleic Acids to compensate for deficits in the endogenous Biosynthesis of these components.

As noted above, Diseases of the aging Organism require specialized therapeutic tactics, since in old age any Diagnosis is intertwined with a range of impairments across all of the body's regulatory systems. Evidently, it is precisely the systemic Nature of the aging process that dictates the preferential use, in such cases, of drugs with a broad spectrum of biological action, which act as non-specific stimulants of fundamental bodily processes: cellular Respiration and proliferation, DNA Damage Repair, hemodynamic correction, the maintenance of immune potential, and the enhancement of memory and psychological status. These are typically extracts from PLANT AND ANIMAL Tissues, compositions of such extracts, and micro-dose supplements of mineral components (Amasiatsi, 1990; McNamara, 1995).

Natural components are incorporated into complex geroprotective preparations whose efficacy has been clinically proven at the Institute of Gerontology of the Academy of Medical Sciences of Ukraine—specifically, Placenta and Spleen extracts, cytotoxic serum, and complex preparations containing Amino Acids and ATP (Korkushko et al., 2002).

Currently, various approaches exist for developing geroprotective agents. A significant portion of these consist of antioxidants, immunomodulators, and antidiabetic drugs. Their efficacy has been confirmed by experimental studies demonstrating that these agents can extend not only the mean, but also the maximum lifespan of animals.

However, modern advancements in analytical biochemistry and Peptide Synthesis, on the one hand, and in Introduction/32.html">Genetic Engineering on the other, make it possible to study methods for correcting age-related changes—both normal and pathological—in detail and at THE MOLECULAR LEVEL (Walford et al., 1980). Methods are being developed to produce complex therapeutic agents with established component compositions for specific, particularly tissue-specific, correction of physiological functions (Fedorova et al., 1974; Karpov et al., 1985; Yamskov, Yamskova, 1998).

As shown in previous chapters, regulatory peptides (RPs) are secreted by various Cells and Tissues as endocrine and autocrine carriers of information regarding local Homeostasis. In addition, they are generated through the Limited proteolysis of high-molecular-weight Proteins present in the humoral environment. These relatively low-molecular-weight peptides possess a broad spectrum of biological activity and coordinate the execution of biological functions across different Organs and tissues. RPs include many Peptide Hormones; however, given the Discovery of the Cytology/cytology/65.html">Diffuse Endocrine System, it is currently impossible to draw a clear distinction between The concepts of a "regulatory peptide" and a "peptide hormone."

The Study of the mechanisms of organismal aging has shown that they are linked in a specific manner to the peptide regulation of homeostasis. It is known that during the aging process, the Thymus and Pineal Gland—the central Organs of the immune and endocrine systems—experience a marked decline in peptide synthesis, leading to disruptions in the peptide regulation of homeostasis (Yakovlev et al., 1991; Khavinson et al., 2001a). Therefore, METABOLISM/2.html">THE CONCEPT OF slowing down aging by restoring the body's peptide self-regulation via exogenously sourced RPs is logically, pathogenetically grounded, and highly promising in modern gerontology.

The bioregulatory approach in medicine was pioneered by V. G. Morozov and V. Kh. Khavinson (1983, 1985, 1996). They developed a method for isolating a complex of Polypeptides from two key organs—the thymus and the pineal gland—whose involution primarily drives the acceleration of aging. When introduced into the body, these polypeptides are capable of restoring the functions of these organs and slowing down the rate of organismal aging (Morozov, Khavinson, 1973). Such regulatory peptide complexes were later named cytomedins. They are characterized by a high degree of purification from nucleic acids, CARBOHYDRATES, and Lipids, alongside a virtually complete absence of allergic and side effects when administered via injection (Khavinson et al., 2001c). Experimental studies demonstrated the normalizing effect of epithalamin on melatonin levels (Anisimov et al., 1992), antioxidant system activity (Anisimov et al., 1997), and lifespan as well as the incidence of spontaneous tumors in animals (Anisimov, Khavinson, 1991). Clinical studies have shown that preparations (cytomedins) isolated from various organs and tissues tissue-specifically stimulate tissue regeneration, restore Immunity, and support the endocrine and central nervous systems (Anisimov et al., 1993; Khavinson, Shutak, 2000; Ryzhak et al., 2001). Significant experience has now been accumulated in the clinical geriatric application of epithalamin and thymalin. These drugs have been shown to possess The ability to inhibit The Development of key processes underlying aging and associated age-related pathologies (Khavinson, Morozov, 1991; Anisimov et al., 1994). In cases of age-related immune decline, regulatory peptides of The Immune System and their complex formulations prove to be arguably the only effective means of compensating for lost bodily functions (Yakovlev et al., 1990).

A fundamentally important issue concerns the replacement versus stimulatory nature of cytomedin therapy. Comparing the lifespan of active peptides injected into the bloodstream, which is measured in minutes (Ashmarin, Kamenskaya, 1988), and the duration of a cytomedin treatment course (typically 5–10 days), on the one hand, with the duration of the therapeutic effect (several months or years) on the other, clearly points to the stimulatory nature of this type of therapy. This provides grounds to believe that the exogenous administration of cytomedins temporarily replaces a compromised link in physiological regulation, enabling the body to restore a weakened or lost function and subsequently maintain it independently over a long period. K. V. Sudakov pointed to this possibility, believing that during natural aging in old age, certain functional systems or their individual components are selectively shut down. At the same time, The Emergence of certain compensatory functional systems within the aging organism remains possible (Sudakov, 1984).

In our view, The Mechanism of such an action is primarily based on the ability of cytomedins to restore and maintain Protein Synthesis in the corresponding organ at a level characteristic of a youthful organism. This process restores degraded cellular protein receptors, thereby normalizing Cell sensitivity to other humoral regulators as well. For instance, thymalin—a cytomedin derived from thymic tissue—exerts precisely this type of action on T-lymphocyte receptors (Napalkov et al., 1998; Morozov et al., 2000). The stimulatory effect exerted by cytomedins derived from animal brain tissues (cortexin and epithalamin) on the Development of the Cerebral Cortex AND subcortical structures positions these drugs as highly promising therapeutic agents for neurodegenerative processes (Rybnikov, Zakutsky, 2000; Ryzhak et al., 2001).

Clinical settings have confirmed the efficacy of epithalamin in treating Climacteric myocardiopathy (Komarov et al., 1995), as well as the combined use of epithalamin and thymalin in elderly women presenting with age-related pathologies (Khavinson, Solovyova, 1998). It is also worth highlighting the crucial results obtained from the experimental and clinical application of cytomedins in A number of pathological processes, which resulted not only in the restoration of organ functions, but also in the normalization of tissue structure (Khavinson et al., 2000a).

For more refined correction and preservation of physiological functions within the organism, synthetic RP preparations are employed, manufactured in accordance with the Amino acid sequences of natural RPs (Ivanov, Berlin, 2000). Several approaches to their synthesis have been developed (Andersson et al., 2000). In some cases, hydrophobic amino acid residues are added to the peptide chain to increase the resistance of synthetic regulatory peptide analogs to cellular Endopeptidases (Ashmarin, Karazeeva, 1999).

A fundamentally crucial milestone in the evolution of our concept of peptide bioregulation in aging is the search for substances with precisely defined structures, which will enable a deeper understanding of their MECHANISM OF ACTION and facilitate the creation of pharmaceutical drugs based on them.

These studies were initiated by the creation of a novel immunomodulating drug based on a synthetic peptide—thymogen (Morozov et al., 1997). The bioactive dipeptide was first isolated from thymalin and subsequently synthesized from glutamic acid and Tryptophan. Thymogen has been shown to exert a stimulatory effect on all stages of T-lymphocyte differentiation (from stem cells to cell-mediated immunity effectors), induce the expression of differentiation Antigens on these cells, and normalize the count and ratio of T- and B-lymphocytes in the Blood during pathological states (Morozov et al., 2000b). Notably, thymogen exhibits biological activity analogous to thymalin at significantly lower doses.

However, it must be emphasized that isolating individual peptides and determining their biological activity is an extremely labor-intensive task requiring the protracted evaluation of hundreds of peptides, which is practically unfeasible. In this regard, a fundamentally new approach to the search and synthesis of physiologically active peptides was developed. Based on an Analysis of the rank order of amino acids within complex cytomedin-Class preparations and the discovery of repeating oligopeptide blocks within regulatory proteins, amino acid sequences possessing specific regulatory properties were identified (see Section 1.4.1). In recent years, this approach has enabled the St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the Russian Academy of Medical Sciences to develop and synthesize peptide BIOREGULATORS targeting the functional activity of the thymus, cerebral cortex, pineal gland, retina, Blood Vessels, Heart, and Bronchi. They have been given the collective name of cytogenes.

A comparative Study of the biological activity of cytomedins and cytogenes revealed numerous similar effects when acting on various normal and pathological organs and Tissues of the body. For instance, the peptides vilon and epithalon—designed based on the analysis of thymalin and epithalamin, respectively—promoted increased lifespan and a reduced incidence of spontaneous tumors, which fully aligns with the results of experiments involving complex preparations of the thymus and pineal gland (Anisimov V. N. et al., 2001, 2002b). It is noteworthy that the dosages of cytogenes used in these experiments were typically 1,000 times lower than those studied for cytomedins. Furthermore, cytogenes were shown to possess tissue-specific and geroprotective actions both *in vivo* and *in vitro* (Khavinson, Mylnikov, 2000; Khavinson et al., 2000b; Anisimov V. N. et al., 2002). Investigations into The impact of cytogenes on the morphofunctional characteristics of various organs revealed a tissue-specific protective effect of vilon and epithalon with respect to vascular structural integrity and the normalization of microcirculatory disorders in these organs (Khavinson et al., 2001d). Experimental studies also established that epithalon restores melatonin and cortisol levels and compensates for neuroendocrine regulation disorders in aged monkeys (Khavinson et al., 2001b).

The application of peptide bioregulators makes it possible to exert targeted effects on a diseased organ. In this regard, It is important to note R. V. Petrov's view that regulatory peptides represent a new generation of therapeutic agents that exert a naturally targeted corrective action without side effects (Petrov et al., 2000).

In our view, peptide bioregulators possess the property of controlling Gene Expression and protein synthesis processes within cells. For example, when studying The Effect of a synthetic Liver peptide on protein synthesis kinetics in monolayer cultures of hepatocytes from rats of various ages, the restoration of both the intensity and rhythm of protein synthesis under the Influence of the peptide was observed (Brodsky et al., 2001). Based on these findings, it should be hypothesized that the geroprotective action of short peptides is linked to the restoration of Protein Biosynthesis processes within cells. Our data indicate that peptide bioregulators are also capable of altering genome functional activity across different Stages of the Cell Cycle AND influencing mRNA synthesis processes, which govern The production of specific cellular proteins (Morozov et al., 2000a). This is all the more significant given the decisive role of the genetic apparatus in the Pathogenesis of many diseases (Bochkov et al., 2002).

In complicated psychophysiological conditions of elderly individuals exhibiting a pathological aging phenotype, optimizing the administration routes of peptide preparations is essential. Alongside injectable forms, RP drug formulations for transdermal and oral delivery have been developed (Chien, 1992).

As is known, human Skin possesses not only a well-developed sensory system, but also a capillary transport system for biologically active components. The regulation of transdermal fluxes from the outside through the skin into The Lymphatic system and bloodstream is maintained by the hydrophilic-lipophilic balance between the epidermis and vessel walls. Certain shifts in the nervous and endocrine systems of older individuals lead to alterations in skin physiological functions. Skin moisture content declines, the shedding of the stratum corneum slows down, and the levels of mucopolysaccharides, Cholesterol, and Phospholipids in the epidermis and dermis decrease. Under these conditions, tissue-specific liposomal creams containing RPs serve not only as a vehicle for the targeted delivery of RPs into the body, but also compensate for the reduced moisture and phospholipid content within the dermis (Chien, 1992).

The development of oral formulations of peptide hormones and regulators is based on creating molecular complexes with polymeric carriers that shield the peptide from the acidic environment of gastric juice and the destructive action of gastrointestinal hydrolytic enzymes (Davis, 1990). In this context, both synthetic hydrophilic copolymers (Akala et al., 1998) and plant- or animal-derived Biopolymers—such as Cellulose ethers, dextrans and other carbohydrates, mucopolysaccharides, and RNA and DNA—can be utilized as carrier polymers.

In the Early stages of investigating Selection/9.html">NUCLEIC ACIDS AS therapeutic agents for radiation injuries and age-related brain dysfunctions, the role of peptide impurities—which were tightly bound to the Nucleic Acids and invariably present in the studied nucleic acid preparations—was underestimated (Fedorova et al., 1974). When oral preparations of Yeast RNA not purified of peptides were administered to elderly patients, an improvement in cerebral processes was observed, including the restoration of memory (Cameron et al., 1963). As noted in Chapter 3, nucleic acids and their tissue-specific complexes can be absorbed by cells via endocytosis and penetrate the Cell Nucleus (Glebov, 1987). The subsequent fate of exogenous nucleic acids within The Cell remains incompletely understood, but it can be hypothesized that nucleoprotein complexes, protected from enzymatic degradation in Lysosomes, participate in Chromatin DNA Repair processes.

The Use of nucleic acids as carriers for regulatory peptides in the development of oral drug delivery systems holds particular promise in light of current understanding regarding the role of Transcription factors in the autocrine self-Regulation of Cell populations (Alberts et al., 1994).

The PHYSICOCHEMICAL PROPERTIES OF the model and natural DNA complexes with regulatory Peptides and Proteins studied by us were detailed in Section 3.3. For Practical Application, it was necessary to evaluate their biological activity and tissue Specificity.

Cells of various organs and tissues differ in their set of Membrane Receptors. Along with receptors essential for cell survival across all stages of organismal development, differentiated cells in interphase express tissue-specific receptors responsible for binding specific ligands (see Section 2.2).

Affinity Chromatography methods are widely used to investigate the specific binding of biologically active macromolecules to cellular receptors. The main drawback of conventional affinity sorbents is the low efficiency of specific receptor utilization upon their chemical attachment to a porous inert support, as well as the loss of native conformation and certain physiological functions of receptors due to the chemical modification Procedure. The literature describes a method for producing affinity sorbents based on cell membrane fragments from erythrocytes, lymphocytes, and hepatocytes immobilized within a polyacrylonitrile matrix. These sorbents exhibit high tissue-specific selectivity in binding their natural protein ligands (Grushka et al., 1988; Chernova, Gurevich, 1996). Similar sorbents can be employed to determine the binding selectivity of peptides and DPCs with cell membranes of a specific differentiated tissue.

To investigate the tissue specificity of natural DPCs, we utilized the method of spatial immobilization of brain tissue and prostate cell membranes into a porous polyacrylonitrile polymer matrix (Ryadnova et al., 2001). Given that the Morphology and sorption capacity of the resulting affinity sorbents were not standardized, affinity chromatography served as a method for comparative evaluation of the binding selectivity of natural and model nucleoprotein complexes with cell membrane receptors of the brain tissue and prostate. Selectivity was assessed using the binding coefficient K. In the absence of selective interaction, K = 1.

Table 11 Interaction of peptide preparations and DPCs with an affinity sorbent containing cerebral cortex cell membranes

Table 11 presents the affinity sorbent binding coefficients for insulin, brain and liver cytomedins, their complexes with DNA, and natural brain and liver DPCs (cytamins).

Analysis of the presented data shows that native DNA binds to the affinity sorbent to a certain extent. This can be explained not only by the non-specific binding of DNA to the polymer matrix, but also by potential interactions between DNA and membrane structures. Apparently, such interactions underlie the penetration of DNA macromolecules through cell and nuclear membranes (Kabanov, Kabanov, 1995). When the non-specific protein insulin is incorporated into a complex with DNA, the degree of its binding to cell membranes increases. This provides a clear argument supporting the feasibility of utilizing complexation between non-Nuclear Proteins and DNA to transport such proteins into the cell, and potentially into the cell nucleus.

The cortexin-containing DPC binds to the affinity sorbent more strongly than free DNA. Evidently, this occurs due to selective interactions between the polypeptide component of the complex and membrane receptors. A similar effect is observed for the DNA–hepalin complex. The natural brain DPC (cerebramin) binds to the sorbent more strongly than the model DNA–cortexin nucleoprotein complex. Apparently, the natural DPC contains not only peptides that specifically bind to brain tissue cell membranes and form part of cortexin, but also other regulatory chromatin proteins that likewise interact with receptors. These potentially include acidic Phosphoproteins localized in the cell nucleus and performing regulatory functions (Teng et al., 1971).

Upon incorporation of cortexin and hepalin into a complex with DNA, the binding coefficient of these preparations to cell membranes decreases. The most probable explanation for this fact is that a fraction of the immobilized cell receptors located within the inner Regions of the affinity sorbent granules is sterically less accessible to high-molecular-weight DPCs than to low-molecular-weight peptides.

A comparison of the binding constants of natural nucleoprotein complexes demonstrates that brain DPCs bind to brain cell membranes more than 1.5 times more intensively compared to liver DPCs (hepatamin). The observed difference in the binding selectivity of these preparations is one of the possible mechanisms underlying the tissue-specific effect of DPCs on physiological functions. The difference in binding coefficients between model DPCs comprising tissue-specific peptides and natural DPCs isolated from the same tissue may be related to conformational differences in the DNA molecule within these complexes. Natural DPCs contain native DNA that is already specifically associated with regulatory chromatin peptides, whereas model DPCs contain highly purified fragments of double-stranded calf thymus DNA, which lacks sites for specific binding to brain regulatory peptides. Apparently, the function of such DNA in model DPCs is limited to protecting the peptide components from Enzymatic Hydrolysis.

Similar results were obtained when comparing the binding of DPCs isolated from The Heart, liver, prostate, cerebral cortex, and thymus (coramin, hepatamin, prostalamin, cerebramin, and thymusamin, respectively) to an affinity sorbent containing immobilized cell membrane fragments of prostate tissue. The DPC preparation isolated from the prostate exhibits the maximum binding selectivity toward this sorbent (K = 3.4). Clearly, it contains autocrine regulatory peptides characteristic of the prostate that possess a high affinity for cells of this tissue. Therefore, natural prostate DPC is able to bind more firmly to the membranes of this tissue. The binding coefficients of the remaining investigated DPCs isolated from other tissues are lower by 30–40%.

The tissue-specific biological activity of liver and brain DPCs was investigated in an organotypic culture of spinal ganglia and chick cerebral cortex fragments (Khavinson et al., 1997). Sensitive Neurons in explants of these tissues respond with enhanced neurite outgrowth upon the introduction of ultrafine concentrations of Neurotrophic Factors into the culture (Levi-Montalchini, 1982). The results of biological testing of natural DPCs isolated from the brain and liver indicate a stimulating effect of brain DPCs: neurite outgrowth increases by 40% compared to the control. No stimulating effect on neurite outgrowth was registered for the liver-derived DPC. This finding demonstrates not only the biological activity of brain-derived DPC toward neurons, but also a distinct tissue specificity of natural nucleoprotein complexes.

This characteristic of natural DPCs proves to be a major advantage when developing oral formulations of these preparations for correcting and stimulating the functions of specific organs in geriatric patients. The mechanism of delivering tissue-specific nucleoprotein complexes to target organs can be represented as follows.

The uptake of nucleoprotein complexes across their entire range of molecular weights and tissue origins occurs in the Small Intestine at the brush border surface via endocytosis. Endocytosis, a process characteristic of the majority of cells, has already been discussed in Section 2.2. It represents the adsorption of a Ligand onto the outer surface of The cell membrane, followed by the invagination of this membrane region into the cell interior along with the bound substance, closure of the membrane to form a vesicle, and the pinching off of this vesicle into the cell interior (Glebov, 1987); The final stage is termed ligand internalization (Willeman et al., 1985). The subsequent pathway of the internalized DPC depends on its specificity. Certain cells (for instance, capillary endothelium, intestinal epithelium), upon capturing substances via endocytosis, excrete them via exocytosis toward neighboring cells, including target cells. This phenomenon has been designated as "transcytosis". Thanks to this mechanism, complexes can be transported to target organs for which they are specific, overcoming tissue barriers.

A nucleoprotein complex that has reached the target tissue can be incorporated into mitochondrial structures or into the cell nucleus depending on its binding selectivity toward the membranes of these Organelles. The penetration of cell cycle control proteins through the nuclear envelope into The Nucleus has been demonstrated in vitro (Protein targeting, 1993). A similar mechanism is feasible for the intertissue and Intracellular Transport of DPCs.

Apparently, There is a profound analogy between the cell's reparative mechanisms during the endocytosis of tissue-specific DPCs and apoptotic bodies. As already mentioned, during aging and natural cell degradation (apoptosis), a limited and organized self-destruction of cellular structures takes place (in contrast to necrosis): chromatin and cytoplasmic material divide into compact fragments, these fragments become enveloped by an adjacent portion of the membrane, and the cell disintegrates into so-called apoptotic bodies, which are engulfed by neighboring Cells of the differentiated tissue via endocytosis and partially utilized for the repair of these cells, including chromatin repair. The integration of exogenous DNA into cellular chromatin can occur only in the presence of breaks in the host cell's polynucleotide DNA chain, which are commonly observed in damaged and aging cells (Sjakste, Budylin, 1992). In this case, another advantage of using natural tissue-specific nucleoprotein complexes is that the DNA components of these complexes contain regions homologous to damaged DNA sites in specific differentiated tissues of the mammalian organism, since they are isolated from identical yet young and healthy mammalian tissue.