(An interview with Professor Vladimir Khavinson)

In this interview, the current President of the International Association of Gerontology and Geriatrics, (European Region) and Head of the St. Petersburg Institute of Bioregulation and Gerontology, Professor Vladimir Khavinson, discusses with Phil Micans the role of peptide bioregulators and their ability to create a protective biological reserve for health and aging.

PM: “Professor Khavinson, it’s a privilege to talk with you today about the peptide bioregulators that you have discovered.”

VK: “Thank you Phil, of course this discovery was the result of much research.”

PM: “Indeed, I have read many of your papers and listened to your lectures. But for our readers, could you please describe how it all began?”


VK: “The journey began in the 1980s when I was a medical Colonel in the then Soviet army. The Kremlin was concerned about new American weapons such as a battlefield laser that would blind troops, or the fact that our submariners’ and missile silo operators were being exposed to low levels of radiation for long periods. They wanted answers that would help protect and maintain the troops’ health and indeed reverse various conditions. In fact, because of their jobs, some of these individuals were literally showing signs of premature aging; so we could say we were developing an antiaging medicine.”

PM: “Fascinating and presumably the resources of the State were behind you?”

VK: “Yes they were, and of course it was all a military secret then. I have to say that I don’t believe it would be possible to repeat much of our research today, because then we could virtually ask for what we wanted and it was made available. Today the cost would be very great for pure research- without a known goal.”

PM: “So what did you discover?”

VK: “We discovered and have widely published that short-chain peptides have a very important role. They are a short-cut to protein synthesis, that each gland/ organ uses a very specific peptide that acts like a gene switch; reinvigorating, triggering if you will a biological reserve. Accordingly, we have named them peptide bioregulators. Importantly we have shown that these peptides can be taken by mouth and make their way into the bloodstream.”

PM: “So, by assumption if one was to ingest the peptide bioregulator for the testes, could a man expect more testosterone release, is that the idea?”

VK: “Yes and often there are more functions beside, so in your example there is also increased spermogenesis.”

PM: “I am aware that these peptide bioregulators today have been patented and are registered on the Russian market as food supplements, how many do you have today and what areas do they cover?”

VK: “Regarding the bovine extracted peptides we currently have 17 manufactured on a regular basis; these are peptide extracts from blood vessels, thymus, cartilage, liver, brain, pancreas, pineal, bladder, stomach, eye, testes, heart, ovaries, prostate, adrenals,  muscles and kidneys. Naturally we intend to add more as our research continues.”

PM: “Have any of these in particular proved to be outstanding in anyway?”

VK: “The pineal extract has had many outstanding results as can be seen in our publications, but all peptides to date have been effective.”

PM: “I know that the dosing of these peptides is quite unique, for example they don’t have to be taken every day. What do you normally recommend?”

VK: “Of course it depends on the need, but the biological reserve is created after a cycled dose and can be maintained for some time thereafter. In nearly every case the biological reserve is improved by 42%. Some individuals who are healthy and wish to maintain health can use as little as 2 capsules daily for 10-days and repeat it 6-months later. Others who require more than just maintenance may need 2 capsules daily for 30-days and repeat bi-monthly, however a lot of patients find their stabilizing dose between those two regimes.”

PM: “Have you used peptides in combination and found any synergistic effects?”

VK: “We have recently designed up to three peptides for various disorders to make these synergistic combinations. In all cases, the addition of the vascular peptide - Ventfort® - is a useful addition; obviously by improving blood supply this can have wide benefits.”

PM: “That’s very useful; IAS will print off that recommended combination list and include it with this interview. Clearly Professor, you have been involved in this fascinating project for many decades, in all that time how many patients do you think it has been applied to and what side effects or contraindications have there been?”

VK: “There are no known side-effects, or contraindications and our methods have been applied millions of times in tens of thousands of patients over decades.”

PM: “What are your thoughts today about these peptide bioregulators and where they might take the future of medicine and health?”

VK: “I think it may improve everyday life of seniors, who constitute the most vulnerable group of population. Europeans and America are getting older and living longer than ever before, nearly 10 years more than in 1960. Increased longevity is a great achievement and a great challenge. It is our task to turn challenges into opportunities and to make the most of the chances offered by the scientific community.  Peptides will bring medicine and health systems to a new level with an accent to preventive medicine which will help to enhance human vital resource and add life to years.”

PM: “Professor Khavinson thank you very much for your insight into this remarkable research. I firmly believe that peptides are going to become a major focus in our field. Congratulations on your intriguing research.”

Introducing The Peptide Revolution - A video about the development of Peptides during the cold war. Russian leadership noted 'Premature Aging Syndrome' in personnel working in certain dangerous conditions and provided the resources for Professors Khavinson and Anisimov to make groundbreaking discoveries. Today Peptides are routinely sold over the counter in Russia and it has taken the West many years to understand and take note of it's interesting properties and effects of the Peptide Revolution.

What tests have been conducted?

Clinical trials and examinations have been conducted at the St. Petersburg Biogerontology Institute and they have concluded that short chain peptides, when applied to skin cells, have many beneficial activities, shown below are some of those results:

Figure 1: A 68 year old female before (left) and after (right) application of Youth Gems®.

How is each Youth Gem® application used?

• Body milk: The body milk is a very light cream that can be applied to most areas of the body, i.e. neck, arms and legs etc. It is designed to help fight against the effects of the loss of skin elasticity with age, providing- after regular applications, a smoother and more efficient skin texture and composition.
• Day cream: The day cream is the core product designed to be applied to the face and hands. Its unique formula provides resilience against skin aging, helping to improve blood supply for a youthful glow and to create a more elastic, compliant skin condition.
• Serum: The serum is designed to be used sparingly against the most noticeable skin aging effects on the face and neck, i.e. applied directly to the fine lines surrounding the eyes and the mouth and chin etc. Regular use will plump up those affected zones and aid in a fuller, more youthful appearance.
• Tonic: This cleanser can be used to help any area become more firm and taught and may be splashed on as required; in particular it makes an excellent after-shave product and helps to prevent irregularities such as acne.

Those who are already familiar with the work of Professor Vladimir Khavinson from the St. Petersburg Biogerontology Institute in Russia, will know how remarkable his work has been in the discovery of short-chain peptide bioregulators- located in food- that act as highly specific gene switches.

Now, four of these peptide bioregulators have been combined into topical skin preparations so that their performance can be brought to the field of aesthetic medicine; specifically the peptides in use are those from:

• Cartilage
• Blood vessels
• Thymus gland
• Pineal gland

What does each peptide provide for?

The beauty product line Youth Gems® contains these peptides and a ginseng extract called Neovitin®. They are a specially developed program of complex skin care designed for the face, neck, hands and the body. The line includes four unique active ingredients of short-chain peptides that have a directed tissue-specific action to improve all basic skin structures, plus a ginseng biomass extract with strong antioxidant and anti-inflammatory action and a number of other natural and synergistic ingredients.

• The thymus gland peptide stimulates tissue regeneration and the synthesis of tissue-specific proteins. Thus, cells proliferative and metabolic activity is enhanced- accelerating the renewal of various cell tissues. It also has an anti-inflammatory action, improving the healing time of wounds, as well as antioxidant, immune stimulating and anti-stress actions.
• The pineal gland peptide regulates metabolic processes and increases protein synthesis in skin cells. It also possesses potent antioxidant activity, normalizes the lipid peroxidation processes in skin cells that in turn promotes the elimination of negative influences on the skin from external factors, (i.e., air pollution, intense sunlight, intensive physical activities and traumas) as well as internal, (i.e., emotional stress) adverse factors. The end result is that metabolic processes in skin tissues become more active and their restoration process is accelerated.
• The cartilaginous tissue peptide stimulates regeneration of fibroblasts and keratinocytes and interferes with destructive changes in collagen skin structure; it also strengthens collagen structure of elastic skin fibers and increases its elasticity.
• The vessel peptide regulates metabolic processes in the vascular wall, normalizes vascular tone, restores disturbed skin microcirculation, (even under the action of adverse environmental factors and emotional stress), strengthens and regulates the permeability of the vascular walls of skin vessels and improves skin turgor.

What else is included in Youth Gems® in addition to the four peptides?

In addition to the four peptides, the Youth Gems also contain an incredible array of beneficial natural agents- which just by themselves would make other antiaging creams jealous! The range includes:

• The bio-antioxidant complex Neovitin® which is a complex of active ingredients isolated from ginseng. It participates in reducing the oxidation processes and therefore acts as a highly efficient antioxidant. Overall, it strengthens the walls of blood vessels, normalizes fat and protein metabolism in skin cells and optimizes the content of micro and macro elements. It is intended to help balance and correct the antioxidant skin status which is very important for the prevention of natural and premature aging of skin and protects against oxidizing stress caused by adverse ecological conditions, smoking and the administration of antibiotics, as well as intense physical and emotional loads, or staying inside air conditioned rooms.
• Furthermore, the bio-complex of cellular membranes isolated from ginseng act to help maintain the integrity of the structures of the cell periphery, keeping its shape and protecting its protoplast. This helps to improve skin elasticity and suppleness making the skin soft and silky.
• Olive oil consists mainly of oleic acid triglycerides with the remainder being glycerides of palmic and stearin acids, cholesterol, lecithin and phytosterol. It makes skin tender and velvety by regenerating and moisturizing irritated and dry skin.
• Raisin-seed oil has anti-inflammatory, moisturizing and regenerative effects. It promotes the peeling-off of keratinized cells; accordingly it considerably improves skin tone and structure. It also helps to regulate sebaceous glands activity and prevent the dilation of pores.
• Argon oil, (obtained by a cold pressing method) is one of the rarest and most expensive vegetable oils in the world. Argon oil contains a lot of vitamin E and vitamin F (non-saturated fatty acids). Argon oil is suitable for any type of skin and is especially effective for treatment of age-related changes such as skin wrinkles, flabbiness or dryness. Its oil is quickly absorbed and gets into the deep layers of the epidermis, improving derma cell condition and promoting the division of healthy cells. It also aids moisturization and nourishment whilst softening the skin, making it elastic and supple.
• Soya oil is an important source of lecithin, with linoleic acid, oleine acid, palmitin and stearin acids. As with all oils rich with irreplaceable fat acids, lecithin and phytosterol, it restores the epidermal barrier and water-retaining ability of the skin.
• Jojoba oil is a mix of non-saturated ether fat that possesses high penetrating ability and is deeply absorbed into the skin providing for the regeneration and protection of deep epidermis layers. Jojoba oil promotes moisture preservation in skin tissues, eliminates peeling and irritation and heals cracks.
• Bisabolol is an extract from chamomile and lavender essence possessing anti-inflammatory and soothing actions.
• Peony extract is an essence which includes salicylic and benzoic acids, tannins, glycosides, iron, chrome, copper, sodium, potassium and other microelements. It is used in beauty products in order to restore epidermis functions.
• Sodium hyaluronate is a derivative of hyaluronic acids that can normalize water balance. It is well tolerated by the skin and is used mainly to correct hyaluronic acid deficiency whose biological synthesis may be disturbed due to aging.
• Starch synthesized from different plants improves complexion and skin functions due to blood microcirculation improvement, the skin gains a pleasant glossy shine.
• Green tea extract is a natural antioxidant with a high content of polyphenols, its activity as an antioxidant is several times higher than the antioxidant activity of vitamin E. Tea polyphenols also have anti-inflammatory and antibacterial effects and they promote penetration of biologically active substances into the skin. Caffeine, being a part of the extract, promotes improvement of blood microcirculation and skin nutrition, reducing edema. Tannins also help to make skin elastic and the extract is rich in vitamins of C, K and B.
• Glycerine is a component of vegetable fats that absorb moisture from the air and keeps it on the skin, making it soft and elastic.
• Xanthan gum is emulsifier and thickener.
• Cocoa oil contains stearin, palmitinic, oleic and linoleic acids. It has healing and toning effects.
• Carrageenan from seaweed is known to reduce allergic reactions.
• Glucose actively participates in salt-water and carbohydrate metabolism, providing the skin cells with energy.
• An active yeast complex promotes the prevention of baggy skin and enhances blood circulation to the capillaries located directly under the skin. It increases the elasticity of skin blood vessels and stimulates the production of vascular endothelial growth factor- reducing black eye-rims and edema below the eyes.
• Fresh water sponge extract possesses anti-inflammatory, anti-edematous and transdermal actions that considerably strengthen the activity of the natural ingredients within the Youth Gems®.
• Sorbitol is found in rowanberry juice and is used as a moisturizing component. It influences serum texture, making it soft, plastic and velvet like.
• Liposomes are hollow microscopic spheres that contain a stable form of vitamin C and rutin which slow down the skin aging process. Liposomes deliver active substances deep within the epidermis layers.
• Panthenol (vitamin В5) possesses healing, moisturizing and smoothing actions.
• Vitamin E is a natural antioxidant that protects the skin against various oxidizing changes. It participates in biosynthesis of proteins, cells respiration and in other major processes of cellular metabolism and protects skin cells against external adverse atmospheric influences.
• Sweet almond extract possesses smoothing and remodelling actions. It forms a continuous, elastic and smooth film on the skin, making it elastic. It also soothes skin and reduces wrinkles.
• Winter bloom has a cleansing effect; it soothes superficial skin layers and promotes the shrinkage of pores.
• Meadow foam oil possesses strong anti-oxidizing action; it moisturizes the skin, creating an invisible protective film on the skin surface, helping to reduce moisture evaporation, thereby improving the skin structure.

Ingredients:

 Aqua, Sorbitol, Glycerin, Panax Ginseng Cell Culture Extract (Neovitin®), Xanthan Gum, Chondrus Crispus Extract, Glucose, PEG-40 Hydrogenated Castor Oil, Propylene Glycol, Prunus Amygdalus Dulcis Seed Extract, Phenoxyethanol, Ethylhexylglycerin, Panthenol, Disodium Rutinyl Disulfate, Hydroxyproline, Magnesium Ascorbyl Phosphate, Tocopherol, Ascorbyl Palmitate, Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Benzisothiazolinone, Methylisothiazolinone, Argania Spinosa Kernel Oil, Citric Acid, Parfum, Thymus, Cartilage, Vessels and Pineal Gland Peptide Complexes, Hexyl Cinnamal.

Action:

With the help of thymus, vessels, cartilage and pineal peptides, plus the bio-antioxidant complex Neovitin®, the Youth Gems® serum intensifies metabolic processes in skin cells and improves its structure. It possesses effective anti-inflammatory action due to the presence of the thymus peptide and Neovitin®. Natural antioxidants within the serum effectively neutralize the negative action on the skin from free radicals, promoting a delay of skin aging. The cartilage peptide promotes normalization of collagen synthesis of skin cells leading to a smoothing of small wrinkles, improving skin micro-relief. The result is intensified by the active ingredients in Neovitin® and the formation of an elastic smooth film, under the influence of sweet almond extract, creates a ‘face lifting’ effect. The serum improves microcirculation in the skin with the help of the vessels peptide, optimizing the metabolic processes of the cells in the vascular wall and together with Neovitin®, the liposomes, vitamin C and rutin, they deliver active ingredients deep into the skin, thus improving skin nourishment and turgor. Youth Gems® serum intensively moisturizes the skin with the help of its specially developed moisturizing complex which is completed with a softening action.

Ingredients:

Aqua, Vitis Vinifera Seed Oil, Corn Starch Modified, PEG-2 Stearate, Ceteareth-25, Cetyl Alcohol, Hydrogenated Coconut Oil, Mineral Oil, Cyclomethicone, Panax Ginseng Cell Culture Extract (Neovitin®), Glycerin, Propylene Glycol, Phenoxyethanol, Ethylhexylglycerin, Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Benzisothiazolinone, Methylisothiazolinone, Butylated Hydroxytoluene, Acrylates / С10-30 Alkyl Acrylate Crosspolymer, Triethanolamine, Argania Spinosa Kernel Oil, Parfum, Sodium Hyaluronate, Thymus, Cartilage, Vessels and Pineal Gland Peptide Complexes, Hexyl Cinnamal, Limonene, Butylphenyl Methylpropional, Linalool.

Action:

With the help of thymus peptides, vessels peptides, peptides of cartilages and the pineal gland, plus the bio-antioxidant complex Neovitin® the Youth Gem® day cream strengthens the metabolic processes in skin cells and improves its structure. The antioxidant action of thymus, vessels, cartilage and pineal peptides, plus the bio-antioxidant complex Neovitin® with argon oil prevents skin aging, making it more resistant to any negative influences from aggressive environmental factors. The action of cartilage peptide, Neovitin® and argon oil promotes smoothing of small wrinkles and softens skin, increasing its elasticity. The cream intensifies and tones up the skin under influence of vessels and cartilage peptides, plus Neovitin® improves the skin’s microcirculation and makes the skin soft and velvet again. It intensively moisturizes the skin with the help of argon oil, raisin-seed oil and sodium hyaluronate.

Ingredients:

Aqua, Propylene Glycol, Glycerin, Panax Ginseng Cell Culture Extract (Neovitin®), Hamamelis Virginiana Water, Phenoxyethanol, Ethylhexylglycerin, Panthenol, PEG-75 Meadowfoam Oil, PEG-40 Hydrogenated Castor Oil, Benzisothiazolinone, Methylisothiazolinone, Parfum, Thymus, Cartilage, Vessels and Pineal Gland Peptide Complexes, Hexyl Cinnamal.

Action:

Youth Gems® tonic is a light liquid with a pleasant smell that effectively and carefully cleanses and soothes facial skin, the skin of neck and decollate against any pollution and make-up, without drying up it. It actively moisturizes and freshens the skin, making it bright looking again, plus more elastic and silky. It possesses anti-inflammatory action and helps to prevent acne. Furthermore, it has expressed rejuvenating effects thanks to action of thymus, vessels, cartilage and pineal peptides, plus the bio-antioxidant complex Neovitin®.

The biologically active peptide bioregulator Cerluten® contains a complex of low-molecular peptides with molecular weight up to 10 000 Da, isolated from CNS of young animals - calves aged up to 12 months.

Cerluten® is manufactured in the form of capsules with a content of active substances of 10 mg.

Experimental studies have shown that the peptides possess tissue-specific action on the cells of the tissues from which they were isolated. Cerluten ® peptides regulate metabolism processes in the brain cells, increase brain safety margins, having a favorable effect on the organism adaptation processes in extreme conditions, the possess anti-oxidative properties, regulating peroxide oxidation processes in the brain cortex. So it is possible to extrapolate an efficiency of administration of Cerluten ® for restoration of the function of the central nervous system and its disorders of various origins.

Treatment of central nervous system diseases is of specific urgency, because they lead to social adaptation disorder and invalidation of patients (2).

Now the treatment of patients with disease of central nervous system diseases in view of pathogenetic mechanisms is carried out using the following traditional therapeutic agents of various action types: (1, 3)

• Influence on metabolism and integrative functions of the brain (cerebrolysin,piracetam, encephalolysate).


• Normalization of cerebral and systemic circulation (stugeron, vinpocetine).


• Reduction of psychopathological signs (meridin, amitriptilin).


• Correction of changes of bioelectric activity of the brain (phenobarbital, convulex).


• Influence on liquorodynamic disorders (veroshpiron, furosemide).


• Prevention and inhibition of adhesive process (aloe, lidasa).


• Correction of immunopathological reactions (levamisole, tavegil).

The clinical studies of Cerluten® were carried out at the Medical Center of the Saint Petersburg Institute of Bioregulation and Gerontology of the Northwest Branch of the Russian Academy of Medical Sciences in 48 patients with various diseases of central nervous system: long-term effects of craniocerebral injury (the prescription of the injuries was from 1 year to 10 years), state after stroke, vascular encephalopathias, decrease in mental capacity, memory, attention during the period from October 2003 till February 2004. Distribution of the patients by clinical entities, sex and age is shown in the table 1. The patients of the main group have been taking Cerluten® in addition to the general purpose medicines - 1-2 capsules, 2-3 times a day before meal for 10-20 days depending on intensity of the pathological process.

The control group consisted of 37 similar patients, which have only general purpose treatment.

All the patients have been taking symptomatic and pathogenetic drugs, these drugs have resulted in short-term therapeutic effect, which required increase in a dose of the drugs per treatment course and duration of their administration.

Conclusion

On the basis of the data received it is justifiably to draw a conclusion that activation of reserve capacity of the brain cortex using Cerluten® helps to improve integral functions of the brain.

Thus the results of clinical study testify the efficiency and expediency of use of Cerluten® for complex treatment and prophylaxis of the diseases of the central nervous system of various origins.

Cerluten® does not result in side effects, complications and drug dependence; there were no contraindications for carrying out of clinical studies.

Cerluten® can be used for treatment and prophylaxis, including in combination with any means of symptomatic therapy, used for neurological practice (vascular, nootropic, resolving, anticonvulsants, vitamins etc.)

Recommendations for use

Cerluten® is recommended for acceleration of restoration of the brain functions after the craniocerebral injury, stroke, intellectual disorders and the influence on the organism of various extreme factors. It’s also indicated to elderly people for maintenance of the mental capacity.

It is recommended to take Cerluten® per os 10-15 minutes before meals 1-2 capsules 2-3 times a day for 10-20 days.

It may be desired to have one more course in 3-6 months.

There are no contraindications and side effects for Cerluten® administration.

References

1. Kovalev G.V. Nootropic medicines. - Volgograd: Nizh.-Volzh. publishing house, 1990. - 368 pages

2. Treatment of nervous diseases: Translated from English/ Edited by V.K.Viderholt. - M.: Medicine, 1984. - 560 pages

3. Mashkovsky M.D. Medicines: Pharmacotherapy for doctors, manual: 2 parts. - Vilnius: ZAO “Gamta”, 1993.


Authors

•    KHAVINSON PROFESSOR, VLADIMIR

The above is an extract of the full article which can be viewed here: 

Introduction

For many years the phenomenon of ageing had been studied within the framework of ethics and social issues. Only in the last century the society realized that the process of ageing should be studied from a different viewpoint, that is, as a special physiologic mechanism bearing a certain evolutionary significance [26,30,67].

Ageing is the most complicate issue in medicine and biology. The process of ageing is a gradual involution of tissues and development of organism malfunctioning. Its symptoms appear already at the end of the reproductive period and become more pronounced with ageing [20].

At the end of the XIX century I. Mechnikov showed that enhancement of cellular immunity contributed to a lifespan increase. He developed a phagocytic theory of immunity and considered human organism capable of combating pathologic ageing [76]. In 1908 he was awarded the Nobel Prize in Physiology or Medicine together with P. Ehrlich. And only one century later, P. Doherty and R. Zinkernagel conducted detailed studies in specificity of cellular immunity in case of viral infection (the Nobel Prize in Physiology or Medicine 1996).

J. Watson and F. Crick jointly with M. Wilkins were awarded the Nobel Prize in Physiology or Medicine in 1962 “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material”.

In 1961 F. Jacob and J. Monod suggested a model of genetic regulation of protein synthesis with the participation of low molecular ligand, which ousts repressor and triggers allosteric conformational transition in the DNA structure of bacterial cell [33]. In 1965 they were awarded the Nobel Prize in Physiology or Medicine along with A. Lwoff. Many years of scientific work by M. Nirenberg and G. Khorana resulted in deciphering genetic code and defining codons (triplets of nucleotides) for each of twenty amino acids (the Nobel Prize in Physiology or Medicine, along with R. Holley, 1968).

Fundamental investigation in nucleic acids biochemistry and identification of RNA and DNA bases sequence were conducted in the 60-ies and 70-ies of the XX century by P. Berg, W. Gilbert and F. Sanger (the Nobel Prize in Chemistry, 1980).

Experimental and clinical studies in gerontology evidenced that immunity of the organism is among the first systemic functions to reveal disorders with ageing [22, 31]. Thymus peptide extracts and peptides, isolated from these extracts, were the first preparations suggested for immune deficit correction [21, 27, 29]. The origin of small regulatory peptides pool in a young organism became evident after the discovery of ubiquitin-mediated protein degradation in proteasomes made by A. Ciechanover, A. Hershko, I. Rose (The Nobel Prize in Chemistry, 2004). Small peptides were shown to play an important role for transmission of biologic information, as for example autocrine hormones and neuropeptides do. A high molecular protein can be hydrolyzed in different ways, degradating into several small peptides. Due to this mechanism there can be produced peptides with completely different biologic functions as compared to the maternal macromolecule [32]. S. Karlin and S. Altschul proved in their works that there are several types of recurring blocks of amino acid residues with charged side chains in protein macromolecules. Nuclear proteins: transcription factors, centromere proteins, and HMG proteins — reliably contain the greatest number of such blocks [34]. Proteasome hydrolysis of these proteins in the nucleus can provide presence of the sufficient amount of peptides with charged side chains.

However, the regulating role of small peptides had never been discussed in the concepts of gene control of protein synthesis in higher organisms before we started the research.

Alongside with immunity decrease associated with age there occur other alterations on cellular level. In particular, there take place changes in the inner structure of cellular nucleus. DNAprotein complex of cellular nucleus (chromatin) gets organized into chromosomes only in case of cell division. In stationary state chromatin is found in two forms: euchromatin and heterochromatin [74]. Heterochromatin is usually localized in the nucleus periphery and contains generally inactive part of genome: genes blocked by repressors. The ratio euchromatin/heterochromatin changes with ageing due to reduction of active euchromatin. This leads to the decrease of protein synthesis in a cell [75].

Thus ageing phase of the organism reveals several levels of dysfunction and may be classified as a systemic syndrome. Promising results of immunodeficiency correction by endogenous regulatory peptides testified the necessity of further enhanced studies [27, 29, 31].

Discovery of peptide regulation of ageing

It is known that specific limit of animal and human lifespan is approximately 30-40% higher than their mean lifespan. It could be referred to the impact of adverse factors causing changes in the gene structure and expression accompanied by disorders in the protein synthesis and organism functioning (Fig. 1).

Specific limit of human lifespan — 110–120 years.

Recent achievements in theoretical and applied gerontology allowed goal-seeking regulation of ageing-associated alterations. Therefore, one of the priorities of gerontology today consists in prevention of premature ageing and age-associated pathology, finally aimed at the increase in an average lifespan, maintaining active longevity and reaching an upper limit of human life [30,51].

The integration of findings made by fundamental research into medicine resulted in a better understanding that clinical progress largely depends on molecular medicine, i.e. investigations conducted in genes and biologically active molecules. The achievements in genetics, molecular and cell biology often underlie molecular medicine in designing new pharmaceuticals and technologies.

The studying of genetic mechanisms of ageing is in the forefront of molecular medicine today. There have been defined

Figure 1

Fig. 1. Potential increase in the average human lifespan up to the specific limit (biological reserve).

genes which regulate mechanisms of individual development and the onset of diseases [67].

The influence produced on gene expression permits correction of cell proliferation and differentiation decreased with age [26, 49]. The study of genetic mechanisms of ageing and development of age-related pathology underlies regulatory therapy — the employment of modulators of transcription, deterring and restoring genetically conditioned alterations occurring with age. It is important to know the genome, occurring disorders and to use substances producing selective effect on gene expression [49].

Designing of effective bioregulators which would contribute to the achievement of special life span limit and maintain major physiological functions constitutes a vital issue in modern biogerontology. Investigations in the problem focus on the role of peptides in prevention of premature ageing [36, 49].

Peptidergic regulation of homeostasis occupies an important place in a complex chain of physiological processes leading to the ageing of cells, tissues, organs and an organism in whole.

Morphological and functional equivalent of ageing consists in the involution of organs and tissues, primarily those referred to major regulatory systems — nervous, endocrine and immune ones. The data available suggest occurrence of age-associated hypoplasia, and in some cases even an atrophy of the pineal gland, thymus, brain cortex neurons and subcortical structures, retina, vascular wall, genital organs [20].

Figure 2

Fig. 3. Expression of transcription proteins (PAX 1) in human thymus epithelial cells (the study was conducted in cooperation with Prince Philippe Biomedical Research Center, Valencia, Spain).

In the 1970-ies we began investigation in the mechanism of immuno-depression both experimentally and clinically. We found out that with ageing there takes place an involution of the central organ of the immune system — thymus (Fig. 2, 3) and that of the neuroendocrine system — pineal gland. We also registered definite decrease of protein synthesis in cells of different organism tissues (Fig. 4).

To restore functions of thymus, pineal gland and other organs we developed a special method for isolation and fractionating of low-molecular peptides from extracts of these organs [24, 25].

On the organism level  we have registered in different animals a significant variety of biologic effects exerted by small peptides especially by peptides of thymus (pharmaceutical “Thymalin”) and pineal gland (pharmaceutical “Epithalamin”) [3, 8, 13, 15, 36, 45, 46, 53, 57].

Figure 4

In numerous experiments, these peptide preparations contributed to a reliable increase in animal mean lifespan up to 25–30% as compared to control [4, 5, 9, 16, 24, 25]. The majority of experiments registered some increase in the maximum life span. The most significant effect in the increase of the maximum life span was noted in CBA mice administered with peptide Ala-Glu-Asp-Gly. It made 42.3% [8]. Of particular importance is a correlation between mean lifespan and the main index of cellular immunity

(reaction of blast-transformation of lymphocytes with phytohaemagglutinin) determining T-lymphocytes function in animals exposed to the effect of thymus and pineal gland preparations (Fig. 5) [9].

A significant increase in the mean lifespan of animals was evidently caused by a reliable anti-tumour activity of low-molecular peptides isolated from thymus and pineal gland which is confirmed throughout 55 experiments during 35 years. The animals manifested a sharp 1,4–7-fold decrease in malignant tumour incidence, both

Figure 5

Fig. 5. Peptide preparations effect on mean lifespan andreactionof blast-transformation of lymphocytes with phytohaemagglutinin in mice.

Figure 6

Fig. 6. Effect of pineal gland preparation on the frequency of tumor incidence in animals.

spontaneous and induced by irradiation or carcinogens (Fig. 6) [5, 9, 10, 16, 87]. It should be emphasized that this unprecedented decrease of tumour incidence was found in the majority of experiments (Table 1). Taking into consideration the general mechanism of carcinogenesis in all mammalians the results of these investigation have a great practical significance for tumour prevention in humans [10, 52].

Small peptides isolated from different organs and tissues as well as their synthesized analogues (di-, tri-, tetrapeptides) revealed a pronounced tissue specific (gene specific) activity both in cell cultures and in young and old experimental animals (Fig. 7).

Peptide tissue specific activity manifested in stimulation of protein synthesis in cells of those organs they had been isolated from. The enhancement of protein synthesis under the effect of peptide has been registered in young and old animals (Fig. 8).

Especially significant appeared restoration of reproductive function in old female rats subjected to the pineal peptide preparation treatment [24]. Thus, estrus phase in animals, analogous to menopause in women, lowered from the initial 95% down to

Table 1

52% after the drug administration, while other phases of the cycle, typical of the normal estrus, increased from the initial 5% up to 48%. Another experiment revealed that none of the old female rats got pregnant after mating with young males. Repeated mating after the administration of the pineal gland peptide preparation entailed pregnancy in 4 out of 16 animals which gave birth to 5–9 healthy off-springs each. Thus there were ascertained main advantages of low-molecular peptides as compared to high-molecular protein regulators: they possess high biological activity, reveal tissue specificity and are

Figure 7

Fig. 7. Peptide tissue-specific (gene-specific) regulation.

neither species specific nor immunogenic. These features make regulatory peptides similar to peptide hormones [1, 4, 18].

A detailed study of molecular weight, chemical properties, amino acid composition and sequence of low-molecular peptides isolated from thymus, pineal gland and other organs had been carried out for many years [77, 78, 79, 80, 81]. The obtained data were used for chemical synthesis of several small peptides. A comparative analysis showed that biological activity of natural and synthetic preparations was largely identical. Thus, for example, thymus dipeptide Glu-Trp stimulated immunity, decreased ageing rate and suppressed occurrence of spontaneous tumours in animals [78, 80].

Biological activity of natural and synthetic peptides appeared to be similar in standard testing in tissue cultures and in animals [11, 12, 17, 42]. These results demonstrate prospects for application of these peptides as geroprotectors [37, 64, 71]. The necessity of searching for new drugs — geroprotectors dictated the onset of preclinical studies of these preparations on different structural levels.

Figure 8

Fig. 8. Effect of the liver and pineal peptide preparations on protein
synthesis intensity in hepatocyte monolayer culture in rats of different age.

On the level of cellular structures, small peptides activate heterochromatin in the cell nuclei in senile patients and facilitate the “release” of genes suppressed as a result of heterochromatinization of chromosome euchromatin areas (Table 2) [48, 74]. Structural condensation of chromatin is in close correlation with functional heterogeneity. Heterochromatinization is known to increase with age and correlate with inactivation of genes which were formerly active [75]. Tightly condensed heterochromatin areas of chromosomes are genetically inactivated and replicate lately.

Decondensed (euchromatin) areas of chromosomes function actively. Active chromatin is obviously a necessary factor for gene transcription activity [74]. As it was said above there are two forms of chromatin in the cellular nucleus: light euchromatin and dense heterochromatin located near nuclear membrane. Gene transcription takes place in the light phase, that’s in euchromatin. The amount of heterochromatin in the nucleus increases with ageing on average from 63% to 80%. Regulatory peptides entail the increase in the

Table 2

amount of euchromatin in the nucleus. This means that more genes become available for transcription factors, and transcription of gene information goes on more intensively as well as protein synthesis.

In other words the more euchromatin there is in the nucleus, the more intensive the protein synthesis goes on in the cell [48, 74, 75].

These experimental results brought us to a conclusion that chromatin heterochromatinization is a reversible process which confirms a possibility of restoring protein synthesis, hence, organism functions [49].

The capability of peptides to induce polypotential cells differentiation is of special significance (Fig. 9) [36]. Thus addition of retinal peptides to polypotential cells of Xenopus laevis early gastrula ectoderm led to the emergence of retinal and pigment epithelium cells. This outstanding result explains a pronounced clinical effect of the preparation of the retina in patients with retinal degenerations [36] and in animals with genetically determined retinitis pigmentosa [59].

Figure 9

Fig. 9. Induction effect of retinal peptides on the polypotent cells of Xenopus laevis early gastrula ectoderm.

Addition of small peptides to ectoderm polypotent cells in the same experimental model led to the onset of various tissues. These experiments showed that peptides are able to induce cell differentiation in regard to the structure of the substance added. The analysis of results obtained gives every ground to conclude that it is quite possible to induce deliberately differentiation of polypotent cells and to use biological reserve of various organs and tissues of the organism contributing to prolongation of life up to species limit.

The number of chromosome aberrations is used as a marker of DNA damages in an ageing organism. Somatic mutations can occur due to accumulation of stable aberrations and underlie age-related pathology, including malignant neoplasia [85]. Reliable antimutagenic and reparative activity of thymus and pineal gland peptides have been confirmed by a reduction in the number of chromosome aberrations in the bone marrow cells and cornea epithelium cells in animals revealing accelerated ageing [14, 19, 49, 69].

On the level of gene activity regulation it was established that administration of peptides Lys-Glu and Ala-Glu-Asp-Gly to transgenic mice caused a 2—3.6-fold suppression of HER-2/neu gene expression (human breast cancer) as compared to the control group. This suppression is accompanied by a reliable reduction of the tumor diameter (Fig. 10) [15, 49].

It was revealed that addition of tetrapeptide Ala-Glu-Asp-Gly to the cultural medium of human lung fibroblasts induces telomerase gene expression and contributes to a 2.4-fold lengthening of telomeres. Activation of gene expression is accompanied by a growing number of cell divisions (by 42.5%), which is the evidence of Hayflick’s limit overcoming (Fig. 11) [40, 41]. This fact fully correlates with earlier stated maximum increase of animal life span (42.3%) after administration of this peptide [8].

Figure 10

Fig. 10. Peptides effect on the development of mammary adenocarcinoma and HER-2/neu oncogene expression in transgenic mice (the study was conducted in cooperation with the National Research Centre on Ageing, Ancona, Italy).

The effect of di- and tetrapeptides Lys-Glu, Glu-Trp, Ala-Glu- Asp-Gly, Ala-Glu-Asp-Pro on the expression of 15 247 murine heart and brain genes before and after peptides administration was studied with the employment of DNA-microarray technology [2].

In this experiment, there were used clones from the library of the National Institute on Aging, USA. This experiment provided unique data on alteration in the expression of different genes under the effect of peptide preparations (Fig. 12). An important conclusion driven from the experiment was that every peptide specifically regulates particular genes. Results of this experiment testify to the existing mechanism of peptide regulation of gene activity. It was also registered that dipeptide Lys-Glu, showing immunomodulating

Figure 11

Fig. 11. Overcoming human somatic cell division limit due to introduction of the peptide into the culture of the pulmonic fibroblasts.

Figure 12

Fig. 12. Peptide effect on gene expression in the heart of mice (the study was conducted in cooperation with the National Institute on Aging, Baltimore, USA).

activity, regulates gene interleukin-2 expression in blood lymphocytes [55].

On the molecular level, there was an obvious gap between multiple evidence of specific effects, caused by regulatory peptides in activation of gene transcription [6, 14, 23, 47, 52, 60, 69, 72, 84], and limited schemes of the process underlying the selective binding of the transcription factor with specific DNA sites. Meanwhile nonspecific binding of proteins with the DNA double helix was proved by physicochemical methods [83]. Activation of gene transcription in cells of higher organisms as a rule needs dozens of macromolecular activators and transcription factors.

We proposed a molecular model of interaction between regulatory peptides and DNA double helix in gene promoter region (Fig. 13, 14, 15, 16) [63, 65].

Geometrical and chemical complementarity of peptide amino acid sequence and DNA nucleotide pairs sequence was assumed as a basis for the molecular model. Regulatory peptide recognizes a specific site in the DNA double helix if its own amino acid sequence is complementary to the DNA nucleotide sequence for a sufficient

Figure 13

Fig. 13. Unfolded peptide Ala-Glu-Asp-Gly conformation (plane projection). There are shown end and side functional groups, capable of complementary interaction with DNA.

length. In other words their interaction is specific due to matching

sequences. Each sequence of the DNA double helix nucleotide pairs forms a unique pattern of functional groups on the surface of the DNA double helix major groove. A peptide in the unfolded - conformation can complimentary fit into the DNA major groove along the double helix axis. We used data on molecular geometry of the DNA double-helix and peptide -thread from scientific

Figure 14

Fig. 14. Metric location of functional groups exposed onto the surface
of the DNA major groove in case of embedding of each nucleotide pai
into DNA double helix. Dash line - perpendicular plane, where
aromatic structures of nucleic bases are located.
—NH2 - proton donors groups;
=7N – proton acceptors groups;
—CH3 – hydrophobic (methyl) group.

publications in order to identify nucleotide pairs sequence for specific binding of the DNA and peptide Ala-Glu-Asp-Gly. The screening conducted showed that this tetrapeptide can be located in the DNA major groove with the ATTTG (or ATTTC) nucleotide sequence on the main chain in compliance with the complementarity of disposition of their functional groups [83].

Figure 15

For experimental testing of the molecular model there were used synthetic preparations: DNA [poly(dA-dT):poly(dA-dT)] (double helix) and tetrapeptide Ala-Glu-Asp-Gly. Gel chromatography helped to prove that peptide Ala-Glu-Asp-Gly forms stable intermolecular complex with the DNA double helix (Fig. 17) [65].

Complementary binding of the peptide with nucleotides sequence on the leading strand TATATA of the double helix can be conducted by six hydrogenous and one hydrophobic bonds between functional groups of both participants.

Figure 16

Fig. 16. Scheme of complementary interaction of tetrapeptide Ala-Glu-Asp-Gly with DNA double helix (“DNA-tetrapeptide” complex on the promoter segment of telomerase gene).

Under normal physiological conditions DNA exists in the form of a double helix two polymeric strands of which are kept together by hydrogenous bindings between pairs of bases of each chain.

Most of the biological processes with DNA participation (transcription, replications) need the double helix to undergo disjunction into separate strands. In particular, it is known, that local separation of double helix strands precedes gene transcription by RNA polymerase. For the transcription onset (synthesis of the matrix RNA) the DNA double helix has to be freed from histones,

Figure 17

Fig. 17. HPLC of peptide and DNA on sefadex G-25 in physiological solution at room temperature.

Figure 18

Fig. 18. The scheme of local separation of strands [poly (dA-dT) : poly (dA-dT)] as a result of peptide Ala-Glu-Asp-Gly binding in DNA double helix major groove.

and in the place where the matrix RNA synthesis starts, the strands of the double helix should be separated (Fig. 18).

Concentration dependent hyperchromic effect (increased optic density 260 nm) was found by spectrophotometery of solutions containing synthetic DNA double helix and tetrapeptide Ala-Glu-Asp-Gly. The hyperchromic effect points out a partial destruction of hydrogen bonds between nucleotide pairs of the double helix and local separation of its strands (allosteric conformational changes) [65].

It was experimentally revealed that separation (melting) of free DNA strands occurs at the temperature +69.5°С. In the “DNAtetrapeptide” system melting of the double helix occurred at the temperature +28°С and was characterized by approximately 2-fold decrease in the values of entropy and enthalpy [66]. This fact points out a thermodynamically simplified way of the DNA strands separation in temperature settings typical of biochemical processes of the majority of living organisms. Thus, DNA strands separation under physiological temperature is not a denaturation but a typical feature of protein synthesis initiation. In vitro experiments show that a small peptide of the definite structure and amino acid sequence can participate in activation of genes transcription on the stage of strands separation in the DNA double helix. Biochemical aspect of this phenomenon consists in similarity of structure and amino acid sequence of a regulatory peptide and a specific segment of the peptide chain of the macromolecular transcription factor.

Thus, the studies of peptides biological activity on different structural levels and of physicochemical processes of their interaction proved an indubitably high physiologic activity of peptide regulators and prospects for their further use. Major conclusion reads that peptides are capable of regulating gene expression. Pre-clinical trials demonstrated high biological activity and safety of synthesized peptides [35, 43, 56, 58].

Thus, the administration of peptides Lys-Glu, Ala-Glu-Asp-Gly to animals contributed to a reduced incidence of tumours and an increase in their mean lifespan [39, 82, 88]. Peptide Ala-Glu-Asp-Pro stimulated nerve regeneration [86], peptide Lys-Glu-Asp-Trp decreased blood glucose level in animals with experimental diabetes mellitus [44], peptide Ala-Glu-Asp increased osseus tissue density, peptide Ala-Glu-Asp-Leu contributed to the restoration of bronchial epithelium cells functions [61], peptide Ala-Glu-Asp-Arg restored myocardic cells functional activity [62].

There are still in progress studies of peptide preparations isolated from the cartilage, testes, liver, vessels, urinary bladder, thyroid, as well as synthesized peptides regulating functions of the brain, retina, immune system, proliferation and differentiation of polypotent cells. These physiologically active substances possess, as a rule, a substantial tissue specific effect and are obviously useful for designing new drugs on their basis applicable in bioregulation therapy [35].

Application of peptide bioregulators in monkeys. Taking into consideration a reliable biological activity of peptides we found it reasonable to study the effect of regulatory peptides in monkeys (Macaca mulatta) [28]. Restoration of the melatonin level in old monkeys (20-26 years old) up to normal (typical for young animals 6-8 years old) following administration of the peptide preparation was among the most significant achievements (Fig. 19) [45].

The same old monkeys revealed restoring to normal indices of a daily rhythm of secretion of the main hormone of adrenal gland — cortisol (Fig. 20). Administration of the peptide or pineal preparation to old animals led also to restoration of glucose tolerance disturbed with ageing. Restoring effect of pineal peptides in respect to the function of the islet apparatus of the pancreas and

Figure 19

Fig. 19. The peptide effect on melatonin productionin monkeys of different age.

Figure 20

Fig. 20. The peptide effect on cortisol production in monkeys of different age (in the morning and in the evening).

glucose metabolism is probably related to the restoration of both β-cells sensitivity to the glucose level in the blood and peripheral tissues to insulin) [45]. Due to full correlation of mechanisms of ageing in primates and humans its seems reasonable to use pineal peptides in order to correct functions of the pineal gland producing melatonin, islet apparatus of the pancreas, and hypothalamushypophysis adrenal system in older people.

Application of peptide bioregulators in humans. Taking into consideration the encouraging data testifying to a high geroprotective activity of both natural tissue specific and synthetic peptide preparations we have been concentrating our attention on studies of geroprotective activity of peptides in old and senile people in recent years [7, 28, 36, 49, 52, 68, 70, 73]. Thus, annual treatment course with thymus and pineal preparations led to a reliable decrease in mortality during the period of observation (6–12 years) (Table 3), due to improvement of brain function and that of immune, endocrine, cardio-vascular systems, increased density

Table 3

Peptide preparation effect on mortality rate in elderly and old patients

of osseous tissue (Fig. 21, 22) [36, 52, 68, 73]. It is noteworthy that application of preparation of the thymus led to a 2-fold decrease in frequency of acute respiratory disease (Fig. 23) [52]. The restoration of melatonin secretion level in patients subjected to administration of preparation of the pineal gland is of special significance (Fig. 24) [68, 73].

Application of the pineal preparation in patients led to significant increase of anti-oxidation activity [3], organism resistance to stress factors, produced normalizing effect on carbohydrate metabolism.

Hypoglycemic action of the pineal preparation was conditioned by the increased insulin secretion

Figure 21

which combined with elevated sensitivity of peripheral tissues to insulin. Modulatory effect of the pineal peptide on the level of glycemia decreased along compensation of the disease. The patients suffering insulin-independent diabetes mellitus complicated by hypertension and treated with this drug revealed reduced arterial pressure and restoration of diastolic function of myocardium [36].

Women suffering climacteric myocardiodystrophy showed a significant therapeutic effect after administration of the pineal preparation which correlated with normalization of their immune and endocrine systems indices [36]. The efficacy of the pineal preparation was registered in treatment of patients suffering aspirin asthma. They showed initially low content of melatonin, as well as patients with asthenia syndrome [36].

Thymus preparation revealed its efficacy in patients subject to thymectomy for thymus tumour. In 6-18 months after surgery, they

Figure 22

Fig. 22. Dynamics of reaction of blast-transformation of lymphocytes with phytohaemagglutinin index in elderly patients in 3 years after 6 courses of peptide preparations.

developed severe immunodeficiency which was evidenced by high incidence of respiratory viral infections, recurrent pneumonia, furunculosis, reduced ability of tissues to regeneration, signs of premature ageing (weakened skin turgor, intense graying, increased mass of fat tissue, disturbed functions of endocrine system, etc.).

These patients were treated with thymus preparation only. Following the course of treatment they revealed restoration of cell immunity, disappearance of furunculosis, increased muscular tonus. Later on there were registered lower incidence of viral infections and pneumonia. The treatment courses were repeated in 6-8 months.

These patients received thymus peptides both of the natural origin (drug “Thymalin”), and the synthesized one (drug “Thymogen”) during 15-20 years. It should be emphasized that application of thymus peptides appeared to be vital for them [36]. Special value of this investigation consisted in identifying correlation of positive results with administration of thymus peptides to thymectomized animals.

Figure 23

Fig. 23. ARD incidence in elderly and old people treated with thymus preparation

The employment of thymus peptide preparations (drugs“Thymalin”, Thymogen”, “Vilon”) appeared efficient in treatment of many diseases and conditions associated with the decrease of cell immunity and phagocytosis: radiotherapy and chemotherapy in cancer patients, acute and chronic inflammatory diseases, massive utilization of antibiotics, in case of suppressed regeneration in posttraumatic period and in cases of various complications, in obliterating diseases of limb arteries, in chronic diseases of the liver, prostate gland, complex treatment of some forms of tuberculosis, leprosy [36].

Peptide preparation “Cortexin”, isolated from brain cortex produces considerable neuroprotection effect. This drug improves memory, stimulates brain repair and accelerates restoration of its functions after stress. The drug is used effectively in case of craniocerebral injury, cerebral circulation, viral and bacterial

Figure 24

Fig. 24. Effect of the pineal gland preparation on melatonin level in elderly people.

neuroinfections, encephalopathy of various genesis, acute and chronic encephalitis and encephalomyelitis. The most pronounced efficiency of the brain peptide preparation is marked in old and senile patients [36].

Peptide preparation “Retinalamin” isolated from the animal eye retina shows vivid clinical efficacy. This unique preparation was created for the first time in medical practice and used in patients with various degenerative diseases of the retina, including diabetic retinopathy, involution dystrophy, pigmented degeneration of the retina, etc. The ability of the drug to restore electric activity of the retina correlated with the improvement of vision and appeared to be its great value [36].

A distinct effect was registered in patients after administration of a peptide preparation “Prostatilen” (“Samprost”), isolated from the prostate gland of animals. The drug proved to be effective in chronic prostatitis, adenoma, post-surgery complications and other age-associated urogenital dysfunctions.

Long-term studies and application of peptide preparations of the pineal gland, thymus, brain, retina of the eye, prostate proved their high therapeutic effect in patients of various age, however their strongest effect was observed in patients over 60. An obvious advantage of these peptide bioregulators-geroprotectors is an absence of any side effects. It is noteworthy that over 15 mln. people with different pathologies were treated with peptide preparations during the period of 26 years. Therapeutic effect was equal to 75-85% on average.

The results of clinical studies obviously prompt prospects for tackling demographic issues [51, 64].

Conclusion The mechanisms of ageing studies showed that an involution of the main organs and tissues of the organism accompanied by a decrease of protein synthesis in cells underlies the process of ageing. Peptide preparations isolated from organs of young animals when introduced into an old organism are capable of stimulating protein synthesis, followed by restoration of the main functions. Long-term application of peptides, both isolated from organs and synthesized from amino acid, was registered to lead to a reliable increase in animal mean lifespan by 20—40% i.e. up to their

specific limit (when peptide application started as a rule from the second half of their life). Small peptides (di-, tri- and tetrapeptides) revealed capability of complementary interaction with the DNA specific binding site on the promoter segment of genes, inducing separation of double helix strands and RNA polymerase activation.

Discovery of the phenomenon of peptide activation of gene transcription points out the natural mechanism of organism to maintain physiologic functions, which is based on the complementary interaction of the DNA and regulatory peptides.

This process is fundamental for the development and functioning of the living substance (Fig. 25, 26). Experimental data obtained

Figure 25

Fig. 25. The role of peptides in the cycle of DNA, RNA and protein biosynthesis.

serve as a confirmation hereto. Peptide incubation with the DNA leads to separation of its strands at 28°C and is accompanied with twice less values of enthalpy and entropy. Activation of telomerase gene expression was reached by incubation with the same peptide at temperature 30° C, and was accompanied by the increased fibroblasts divisions by 42.5%. Administration of this peptide to animals resulted in an utmost increase in their mean life span by 42.3%, which correlated with the phenomenon of increased division of fibroblasts.

Application of peptide bioregulators in humans for preventive purposes led to a significant rehabilitation of the main physiological functions and a reliable mortality decrease in different age groups during the period of 6—12 years.

It should be emphasized that this approach to the prevention of ageing is based not only on experimental and clinical data, but also

Figure 26

A report of the results of clinical study of the biologically active peptide bioregulator of blood vessel origin:

The biologically active peptide bioregulator Ventfort® contains a complex of low-molecular peptides with molecular weight up to 10000 Da, isolated from the vascular (aorta) tissue of young animals - calves aged up to 12 months.

Ventfort® is manufactures in the form of tablets or capsules with a content of active substances 10 mg.

Experimental studies have shown that the peptides possess tissue-specific action on the cells of the tissues from which they were isolated.  They improve trophism of the vascular wall cells and provide regulating action on the metabolic processes in them, provide normalization of functional and morphological changes in the vascular wall, they regulate blood content of cholesterol and lipoproteins, thus decreasing a risk of various vascular defects.  So it is possible to extrapolate an efficiency of use of Ventfort® for recovery of vascular function at various diseases, including vascular atherosclerosis.

Atherosclerosis and its consequences is one of the main causes of invalidation and death in developed countries.   Age related changes of vascular wall and hemodynamic disorders results in decrease of peripheral blood flow, vascularization of bodies and tissues, development of various components of oxygen insufficiency and trophesies. (2, 3, 4, 6).

Drug treatment of atherosclerosis is aimed at normalization of lipid metabolism, blood coagulation processes and metabolism in the vascular walls (1, 5).

§  Medicines, which normalize cholesterol and b-lipoproteins level.

§  Those drugs which prevent cholesterol absorption in intestines, (cholestiramin, b-sitosterol, diosponine, polysponine).

§  Drugs which affect cholesterol synthesis in an organism, (clofibrate, miscleron, regardin, cetamifen, nicotinic acid, vitamin PP);

§  Those drugs which increase disintegration and excretion of cholesterol from an organism (linetol, arachiden).

§  Others that improve microcirculation and normalize vascular permeability, decrease edema of vascular tissues and improving metabolic processes in the vascular walls, (prodectine, dicynon, doxium, glivenol, escusane etc.)

The clinical studies of Ventfort® were carried out at the Medical Center of the Saint Petersburg Institute of Bioregulation and Gerontology in patients with atherosclerosis of various arteries and senile purpura during the period from November 2003 till February 2004.

Clinical characteristic of the patients

The subjects of the clinical studies were 49 patients with arterial atherosclerosis and senile purpura, 27 of them were the main group (15 men and 12 women) - they were additionally prescribed Ventfort® per os 10-15 minutes before meal 1-2 capsules 2-3 times a day for 10-15 days depending on a degree of pathological process. 22 patients (11 men and 11 women) in the control group have been taking only general purpose medicines.  The age of both groups of patients has made from 52 up to 84 years old (Table 1).

The patients of both groups have various clinical signs depending on affection of various caliber vessels: essential hypertension, ischemic heart disease, cerebrovascular disorders with memory impairment, clouded sensorium and affective liability.  All the patients had progressive dynamics of pathogenic pathway.

All the patients have been taking symptomatic and pathogenic therapy for specific clinical signs of vascular pathology.

Table 1

Distribution of the patients by clinical entities, sex and age

Examination methods

The patient complaints were assessed and compared, general clinical studies of blood and urine were carried out together with blood biochemical test using “REFLOTRON” device (Boehringer Mannheim, Germany). Blood coagulogram and tourniquet Hesse testing were carried out with a purpose of homeostasis assessment.

Examination results

It was established that Ventfort® administration in patients with arterial atherosclerosis has resulted in improvement of general state of health, especially in patients with cerebrovascular disorders.

As you can see in the table 2, Ventfort® administration has resulted in reliable decrease in the level of general blood cholesterol. There were also a tendency towards decrease in the content of very little density lipoproteins, being most atherogenic.

The patients with senile purpura after Ventfort® administration had an increase in strength of capillary walls according to the results of Hesse testing, the frequency of hemorrhages has decreased. In most patients the skin and hair condition has improved.

Ventfort® administration has not resulted in any side effects, complications, contraindications and drug dependence.

Ventfort® is convenient for administration in hospital, out-patient conditions and at home.

Ventfort® can be used as medical and preventive mean in the form of biologicall active food additive in the form of adjunct for complex therapy of vascular atherosclerosis and improvement of microcirculation in different tissues in combination with any means of symptomatic and pathogenetic therapy.

Conclusion

The biologically active peptide bioregulator Ventfort® provides regulation of blood cholesterol and lipoproteins contents and promotes improvement of vascular wall condition.

Ventfort® is well tolerated at oral administration, does not have any side effects, does not have any contraindication and can be used as an adjunct to complex treatment and prophylaxis of vascular disorders of various geneses.

Ventfort® is recommended for improvement of the function of vascular wall in case of atherosclerosis, microcirculation disorders in the bodies and tissues art various diseases, influence on the organism of various extreme factors.  It is also recommended to elderly people for maintenance of the vascular system function.

Edited by Prof. Vladimir Kh. Khavinson,

Associate Member of the Russian Academy of Medical Sciences

Written by G.A. Ryzhak

Safety of Natural Peptide Bioregulators. – St. Petersburg: IKF Foliant, 2002. – 20 p.

The proposed scientific publication presents the results of a detailed study on peptide bioregulators isolated from animal organs and tissues and on the risk of their contamination with infectious agents, functionally active protooncogenes, nucleic acids, and prion proteins. The medical application of this class of pharmaceuticals has been proven entirely safe.

Introduction The contemporary epoch is marked by an extraordinary diversity of unfavourable factors affecting the human organism: natural conditions, unbalanced nutrition, environmental factors, external damaging agents including ionising and microwave radiation and toxic substances. It results in the depletion of adaptation and compensation mechanisms, occurrence of various diseases and pathologic states, and, finally, in premature ageing.

The above problems necessitate the development and clinical implementation of new effective therapeutic agents and the methods of functional correction, intensification of resistance to adverse factors, inhibition of ageing, and prolongation of life span.

Long-term experience of applying peptide bioregulators extracted from the organs and tissues of young animals has confirmed the high efficacy of this class of substances in various diseases and pathologic states including those not responsive to the treatment with other medications.

However, the active application of animal-derived peptide bioregulators in medicine requires special control over the quality and safety of these substances, since raw material used in their production can contain components hazardous for the human organism. In this view, it becomes ever more significant to devise technologies securing the safety of such substances and to work out effective control methods proving the absence of infectious agents, protooncogenes, prion proteins or other objectionable components in them.

natural peptide bioregulators The concept of bioregulation therapy based on the pathogenetic application of peptide bioregulators in various diseases, pathologic states, and ageing has been proposed and grounded in the course of 30-years’ research conducted at the Military Medical Academy (St. Petersburg) and St. Petersburg Institute of Bioregulation and Gerontology. Vyacheslav Morozov and Vladimir Khavinson were the first to isolate peptide bioregulators of multicellular systems from the hypothalamus, epiphysis, and vessel walls in 1971 [7]. These substances have been brand-named cytomedins.

The technology of obtaining cytomedins includes acetic extraction of polypeptide fractions from cattle and pig tissues, their precipitation and subsequent multistage purification. The final product is in the form of lyophilised powder for injection solutions.

These developments have enabled the creation of new pharmaceuticals – peptide bioregulators, which form a group of pharmacologically active substances with molecular weight within 1-10 kDa. Their administration to the organism results in the functional restoration of the very organs, which the cytomedins have been isolated from.

The Institute possesses 15 pharmaceuticals. All of them have undergone thorough trials. Some cytomedins have been approved for clinical use over 15 years before (Epithalamin® – the endocrine system bioregulator, Thymalin® – the thymus bioregulator, and Prostatilen® – the prostate bioregulator) [5]. In 1999, the Russian Ministry of Health certified the clinical application of Cortexin® (the cerebral cortex bioregulator) and Retinalamin® (the retina bioregulator) [15, 4]. Other bioregulators are undergoing various stages of clinical and experimental studies.

The technology of bioregulators manufacture and the methods of treatment with their use are covered by 70 licenses and USSR, Russian, and foreign patents. The majority of the developments have no analogues in the world.

Long-term experience of applying peptide bioregulators in healthcare has revealed their high effectiveness in various diseases and pathologic states including those not responsive to the treatment by other therapeutic means.

A new class of parapharmaceuticals – cytamins – has been created to expand the sphere for the application of bioregulators. Cytamins are balanced tissue-specific nucleoprotein complexes isolated from animal organs and tissues [11].

Today, 17 Cytamins are produced: extracted from the brain, liver, prostate, heart, thymus, bronchi, cartilages, pancreas, vessels, stomach, testes, epiphysis, thyroid, adrenal glands, kidneys, ovaries, and eye tissues.

Cytamins exert a targeted (organotropic) effect immediately upon the organs and tissues they have been isolated from. Being not drugs per se they produce a mild physiological regulatory influence on various functional systems of the organism, which enables their use as natural adaptogenes. Cytamins promote optimal functioning and full-value nourishment of organs and tissues, they do not contain any conservatives or other alien substances, produce no side effects, have no contraindications, and are compatible with any other nutrients and medications.

The patented technology of cytamins manufacture includes alkaline hydrolysis from tissue cells, consecutive precipitation of nucleoprotein complexes, their purification from ballast substances, and manufacture of the ready form as enterosoluble tablets or capsules.

Thus, two new classes of peptide bioregulators have been developed: pharmaceuticals cytomedins and parapharmaceuticals cytamins applied on a wide scale in medical practice to prevent various disease and pathologic states and treat for them.

safety of cytomedins and cytamins Historical background of the prion diseases concept

The history of the prion diseases concept is set forth in detail in monograph by V.A. Zuev et al. “Prion diseases in humans and animals” [16]. Below we resort to some excerpts from this book.

The problem of prion diseases arose within the framework of the slow developing infections theory on the basis of the results of long-term investigations on mass diseases in sheep imported to Iceland from Germany in 1933. Despite some obvious clinical differences and uneven localisation of lesions in the animals’ organs and tissues, a principal similarity was revealed. This similarity can be summed up in four major signs of slow developing infections:

• unusually prolonged incubation period (for months and even years);
• slowly progressing course of the disease;
• untypical character of lesions;
• Inevitable lethal outcome.

Among the studied sheep pathologies, scrapie was investigated. This disease of sheep and goats was well known in many countries.

Three years later, in a geographically opposite region – New Guinea – a new disease, now known as kuru, was revealed among Papua cannibals and described. Its mass development pointed at its infectious nature.

These and a number of other diseases were united by a common typical symptom. All of them were manifested only as lesions to the central nervous system: a typical picture of so-called spongiform transformation of the grey and/or white brain substances and, sometimes, of the spinal marrow based upon primary degenerative processes (without inflammation signs) and, in some cases, accompanied by the formation of amyloid plaques and expressed gliosis.

This peculiarity of the pathomorphologic picture marked out the name for the entire group of diseases – transmissible spongiform encephalopathies. Their pathognomonic feature consisted in the transmissibility of spongiform alterations solely within the central nervous system.

For decades, any attempts to discover pathogenes of these diseases ended in a failure though their infectious nature was convincingly confirmed. Yet, in early 80s Stanley Prusiner, an American biochemist from the California University, claimed that this pathogene was a nucleon-free low molecular protein (27-30 kDa), which he named “the infectious prion protein” [13]. Prusiner proposed the term “prion” – an anagram from “proteinaceous infectious (particle)” as an infectious unit consisting of the infectious prion protein molecules. Prusiner was awarded a Nobel Prize in Biology or Medicine in 1997 for his prolonged investigations on slow developing prion infections.

Results of the studies carried out over the recent 15 years confirmed the prion nature of transmissible spongiform encephalopathy pathogenes completely. Consequently, these diseases were defined as prion ones.

The non-infectious (cellular) prion protein is vitally important. It is found in all mammals including humans. Among its distinguishing features is its high sensitivity to the digestive activity of protease K, which destroys this protein.

The infectious prion protein is preserved after the digestive impact of protease K. Its molecular weight equals 27-30 kDa.

The mechanism of accumulating the infectious prion protein in an infected organism has not been defined by now. At the same time, it is obvious that in a healthy organism the infectious prion protein entails the transformation of the normal prion protein into its infectious form due to the conformation changes of the normal prion protein. Therefore, the infectious prion protein is accumulated not via de novo synthesis of its molecules in an infected organism, but due to the conformation alterations of already synthesised normal molecules of a prion protein under the effect of the infectious prion protein.

The modern classification of prion diseases distinguishes human and animal pathologies and includes four human and six animal ones.

The list of human prion pathologies starts with Creutzfeldt-Jacob disease – the main in this group, while kuru and Herstmann-Streussler-Scheinker syndrome are considered to be its variants.

Scrapie is the main animal prion disease viewed as the prototype for all human and animal prion diseases.

In 1986, the United Kingdom was struck by an epizootic of bovine spongiform encephalopathy. As it turned out, the infectious prion protein originated from meat/bone flour. The technological process of its manufacture in early 80s implied a considerably reduced process of carbohydrate dissolving extraction of fat from bones, pluck, and heads of caws and sheep. This technological modification enabled the preservation of the infectious prion protein in raw material and caused mass contamination of ruminants after adding meat/bone flour to their forage as a protein supplement.

Started with two cases in 1986, the epizootic developed rather intensively and reached the peak of 1,000 cases in caws in 1992. Then the rate of new occurrences decreased notably and progressively.

Over this period, 23 sporadic cases of the so-called the new variant of Creutzfeldt-Jacob disease were registered, chiefly in the United Kingdom. The disease broke out among young patients, which was untypical.

Creutzfeldt-Jacob’s is a rare and fatal disease of worldwide distribution. Its total annual incidence in different regions is nearly equal and makes 0.3-1 case per 1,000,000 people. There has been revealed no correlation between Creutzfeldt-Jacob disease and the development of scrapie as the most frequent animal prion pathology. Creutzfeldt-Jacob disease has the same incidence in the scrapie-endemic United Kingdom and in Australia and New Zealand where scrapie has not been registered for years.

Wide-scale research was conducted in the United Kingdom in response to the epizootic of bovine spongiform encephalopathy in this country. Results of these investigations revealed the principal risk of infecting humans with prions from contaminated animals. It must be noted that the epizootic did not influence the total incidence of Creutzfeldt-Jacob disease in the United Kingdom as compared to other countries where no cases of the pathology in cattle were observed. Only the young age of all patients with the new variant of Creutzfeldt-Jacob disease attracts attention: all these people were less than 40 years of age. At the same time, young patients with this diseases constituted as little as 2 % of the total population (usually it was registered in the older age group, its peak striking the 60-65-year-olds).

Consequently, natural raw material of animal origin can potentially include infectious agents, protooncogenes, and nucleic acids. That is why, in the manufacture of preparations from animal organs and tissues a special attention is to be given to the purification of the active substance from objectionable admixtures and, first of all, to the infectious safety of such preparations.

Technological aspects of peptide bioregulators safety

In the manufacture of peptide bioregulators only the Russian-raised cattle is employed – calves and pigs under 12 months of age from the regions and farms where no human-endangering infectious diseases including transmissive bovine spongiform encephalopathy has been registered. It is to emphasise that Russia is known for its epizootological and epidemiological safety in respect to prion diseases.

The technology of obtaining cytomedins implies the acetic extraction of polypeptide fractions from calf and pig tissues, their precipitation and subsequent multistage purification until the outcome of active fractions with the molecular weight of 1-10 kDa. The final product is in the form of lyophilised powder intended for injection solutions.

The technology of extracting Cytamins includes alkaline hydrolysis of tissue cells, subsequent precipitation of nucleic complexes and their purification from ballast substances, drying of the half-product and manufacture of the ready form as enterosoluble tablets or capsules.

Thus, the patented methods of obtaining cytomedins and cytamins secure the maximal protection of these preparations from the interference of infectious agents. The active substance is extracted from animal organs and tissues at 3-7°C, pH 3.0 (cytomedins) or 11.0 (cytamins) for 6 days. These conditions are sufficient to fully inactivate all microbes and viruses even if no extra impacts are applied [8, 10].

Analysis of peptide bioregulators molecular weight

Molecular weight of cytomedins was studied by two methods – gel chromatography in Sefadex G-25 and electrophoresis in 15 % polyacrylamide gel. The results of gel chromatography revealed one peak in each chromatograms of the Cytomedins in the region of molecular weight under 10 kDa (Figures 1, 2, 3).