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By Yevgeni PANARIN, RAS Corresponding Member, Director of the RAS Institute of High Molecular Compounds (St. Petersburg); Nina KUZNETSOVA, Dr. Sc. (Chem.), senior researcher of the same Institute.
Progress of high-molecular compounds chemistry and pressing problems of medicine brought about in the late 1950s the emergence of a new field of science- chemistry of biomedical polymers. It deals with the synthesis and study of the properties of water-soluble agents which have certain medicinal functions.
Within this country the field was spearheaded by USSR Academy of Sciences Corresponding Member Sergei Ushakov (1893 - 1964) who assumed that physiologically active substances remain active m combinations with water-soluble polymers. That has opened up the prospects of producing new and more effective medications. The fact is that macromolecules as such have large dimensions and are slowly evacuated from the organism through kidneys; therefore low-molecular medications linked with them take longer to pass through the bloodstream. By that token the duration of their effect is prolonged, while the dosage and the number of injections is decreased. There is also a chance of high local concentrations of preparations in the target organ, their pharmacokinetics undergoing changes and toxicity drastically decreasing and so forth.
The task was manifold. The first problem consisted in seeking and developing proper water-soluble carrier-polymers which in contact with the organism would not cause negative side effects, would not accumulate in the organism and would be disposed of on the completion of their transport functions. The disposal of macromolecules is only possible if their size does not exceed that of kidney uriniferous tubules pores. The problem was primarily resolved through the synthesis of macromolecules with the target mass and narrow size distribution (VP, ethenol, propenamide polymers, etc.) capable of degrading with the production of low-molecular fragments easily disposed by the organism. In the latter case polymers are synthesized containing instable groups in the main chain (polyesters, polypeptides), or natural proteins are used (hemoglobin, albumin, etc.).
Simultaneously, the development of base synthesis methods was in progress, containing a broad set of the required various reactive functional groups. Those served as anchors of sorts hooking up medication substances to a macromolecule. Since in certain cases, to display activity, the latter have to "unhook" medications from the macromolecule in the required place, e.g., inside the cell, such anchor should have complex chemical structure and contain both enzyme and hydrolytically instable groups and chemical linkages. Therefore, chemical construction of polymeric medications poses a complex and challenging task.
Preliminary biological studies of polymeric medications undertaken in the process of experiments on animals
in Leningrad medical institutions have confirmed that the key postulations formulated by Dr. Ushakov are correct. That has laid the basis for deploying studies in our city, primarily concentrated in our institute. Experimental pre-clinical and clinical study of polymeric medications and preparations has involved a large number of research and academical medical institutions of Leningrad.
As a result, by the early 1970s three main research tasks were defined: modification of known medications with synthetic, natural and biosynthetic polymers to improve their therapeutic properties and create new preparations; synthesis of functional biologically active polymers and linking chemical structure to activation; development of polymers for the production of plasma and blood substitutes with improved hemodynamic properties, including oxygen carriers.
As a follow-up of the above effort, studies were undertaken related to the modification of biologically active and medical substances with synthetic polymers- antiphthisic, anaesthetic, antipyretic, anticoagulants, antibiotics and so forth. That has culminated in the development of antiphthisic preparations "Sovinizon" (on the basis of isonicotinic acid hydrazide) and "Sovinax" (based on paraaminosalicylic acid), which possess low toxicity and prolonged action and which have demonstrated high therapeutic effectiveness during tests. Experts of the PLASTOPOLYMER R&D Association have developed what they call experimental-industrial manufacturing technology of the new substance.
One should also note successful studies of complexes of iodine with polyvinyl alcohol. These compounds possess a broad antimicrobic spectrum without a burning effect on tissue like that produced by iodine. Experts have developed tixotropic gels with a high curative effect and preparation "lodinol" which is still produced by our pharmaceutical industry.
And let us stress at this point that a transformation of a low-molecular substance into a macromolecular form not only serves to prolong its "life" and results in some new and unexpected effects caused by high local concentrations of the drug and its "cooperative" interaction with cells and other targets and also con-formational transformations of macro-molecules. The result is an increased specific activity and lower toxicity of preparations which makes it possible in many cases to substantially broaden the range of harmless concentrations, extend the area of medicinal applications and build-up a "depot" in the necessary organ.
What is more, a macromolecular form of polymer makes it possible to include into its structure substances with different mechanisms of action and thus obtain systems with polyfunctional biological activity.
This has been most vividly demonstrated in studies of polymer derivatives of antiseptics from the range of cationic surface-active substances, glucocor-ticoidal hormones and inhibitors of certain ferments.
Glucocorticoidal hormones-hydrocortisone, prednisolone, dexamethasone and others- are well known as anti-inflammatory drugs. Their application, however, causes some negative effects like immunodepression, suppression of the functions of the adrenal glands, timus. And prolonged use of glucocorticoids can cause atrophy of these organs. This is especially dangerous in pediatrics because it is always accompanied by a retardation of the physical development of children due to the arrest of the biosynthesis of endogenic hormones.
And the inclusion of glucocorticoidal hormones into hydrophilic macromolecules has made it possible to obtain their water-soluble derivatives which interact with target- cells without entering them and thus retain their strong anti-inflammatory and antishock properties without any negative side effects.
In our further work with polymers it was established that they are not evenly distributed across the organism, but can accumulate in some particular organ depending on the chemical structure. Where and in what amounts depends above all on the presence in a macromolecule of positive or negative charges, specific lig-ands or vectors. And that means that by varying the chemical structure of a polymer and introducing into it some specific functional groups or fractions of biomolecules one can implement "targeted" drug delivery. For example, in studies of a range of copolymers of vinylamine we discovered their ability, after peroral administration, to become localized and remain for several days on the walls of the large intestine. This fact opens up a perspective of producing peroral preparations of prolonged action which will make it possible to give up injections and develop simple and easy-to-use medicines. In that manner we produced "Poglucar" preparation for the prevention of tumors of urinary bladder. The mechanism of its action is based on the suppression of activity of the enzyme of 3-glucuronidase with the help of a special inhibitor-lacto- saccaric acid. It is interesting to note that in peroral administrations in its pure form the preparation is quickly evacuated from the body (in 2 - 3 h) and because of
Modification of cationic surface-active substances by polymer ("Katapol").
that attempts at its clinical uses for preventing relapses of cancer of the urinary bladder after surgery were of no avail. And linking lactosaccaric acid with the polymer produced a long-lasting inhibiting effect. The "Poglucar" has now passed clinical tests and has been cleared for use and put on the list of drugs permitted for use in Russia.
As has been mentioned, medical uses of a biological active substance can be obstructed by its increased toxicity or other undesirable nuances. Say, cationic surface-active substances have abroad range of antibacterial properties which accounts for their use as disinfectants. But their uses as medicinal drugs is obstructed by their high toxicity and skin-irritating action. But if these substances are modified by copolymers of vinyl-pyrrolydone, their excessive toxicity is reduced by 1.5 - 2 times, skin-irritation effect-by 10 and antimicrobial activity by 3 to 4 times. And that means that, in a new modified form, standard antiseptics can be of great interest as effective cures of purulent and fresh wounds, burns and for the disinfection of surgery fields and surgeon's hands and surgical instruments, etc.
This approach has been put into practice in a polymer antiseptic preparation "Katapol" which is cleared for hospital use and is produced by our industry ("Farmakon" Plant, St. Peters-burg). Local applications of "Katapol" in the form of water solutions of 0,5 - 1 percent and its use in combination with antibiotics for the prevention and treatment of wound infection and in patients with open bone fractures, osteomyelitis, burns and purulent wounds of soft tissues can be 2 to 3 times more effective than the use of traditional low-molecular antiseptics.
What one could call widespread "pollution" of surgical clinics with strains of microbes resistant to antibiotics has had a strong negative effect on therapeutic procedures even despite the introduction of new preparations into medical practice. The situation is especially acute in traumatology, surgery and treatment of burns. This accounts for a growing interest over the past few years in preparations with a broad antimicrobial spectrum-alternative antibiotics. A special place among them belongs to preparations incorporating silver. Our own scientists went on with their investigations, finally producing "Poviargol" on the basis of stabilized water-soluble polymers with nanograins of silver (polyvinylpyrrolydon). It features a broad spectrum of antimicrobial action which is especially welcome in traumatology and surgery and in treatment of pyoinflammatory complications (suppuration of postsurgical wounds, osteomyelitis, trophic ulcers, abscesses and phlegmons, arthritises, gas gangrene etc.); in the treatment of heat burns (combustology) and acute frostbites; in otolaryngology and pulmanology (rhinitis, maxillary sinusitisangina, otitis, purulent forms of tracheobronchitis etc.); in stomatology and sanations of mouth cavity; in ophthalmology for the prevention and treatment of purulent conjunctivitis; in urology, obstetrics and gynecology (purulent cystitis, uretritis, infections of urethra etc.). "Poviargol" is cleared for medical use and is applied in the form of water solutions of 1 - 5 percent concentrations. It is produced by the Special R&D Bureau "Tekhnolog" (St. Petersburg). By its antimicrobial properties, curative efficiency and toxicological characteristics this preparation surpasses silver proteinates. And it also possesses anti- inflammatory properties, stimulates reparative processes in damaged tissues and activates cell and humoral immunity.
In the development of polymer medicines it was originally assumed that the polymer had only act as a "transport" while being biologically inert. But further studies proved that preparations carrying a positive or negative charge (polycations or polyan-ions) interact with cell membranes and many natural macromolecules (proteins, nucleic acids), active on the molecular and cellular levels and interact with the organism as a whole. Thus polycations, linking with cell membranes, strongly change their transport properties, boosting the transit of substances both into and out of cells, manifest antimicrobial properties and boost the effect of antibiotics. In recent time these "traits" of polycations have been closely investigated in a bid to use them for DNA transport into cells for solving the problems of gene therapy and genetic engineering. And compounds have already been found which can help solve this problem.
Polymers with only negative charges have been found to possess anti-tumor and anti- viral properties and they promote the production of interferon by the body. The latter property is manifested in particular by bifilar polyribonucleotides-synthetic analogs of nucleic acids.
Detailed studies of biological functions of polymers, in particular their influence upon young animals, have made it possible to reveal a hitherto unknown property of stimulating the growth and development of young stock. The "Doksan M" veterinary preparation has been produced on
Erythrocyte, hemoglobin and polymeric hemoglobin: a -human erythrocyte, 5 - 6 Mm;
b - hemoglobin molecules, ~0.006 Mm;
с - "artificial erythrocyte" model, 1 -2 Mm;
d - polyhemoglobin macromolecules, 0.02 - 0.05 Mm.
this basis which stimulates digestion and the absorbtion of nutrients from the gastrointestinal tract. It is used as a fodder additive for young horned cattle, pigs and sheep and in poultry farming it boosts the egg-laying productivity of hens and their growth rate. The preparation is cleared for unrestricted use in Russia (before, was produced on an experimental scale).
Of particular interest within this context are recently synthesized in this country polymers which boost stress resistance of animals.
Finally, about the problem of man-made blood substitutes which has been accentuated by shortages of donor blood, its short conservation period and the danger of viral infections.
Research is proceeding in two directions: development of new, and improvements of plasma-substitute solutions which can "imitate", for example, the haemodynamic properties of plasma; development of blood substitutes involved in the central respiratory function-supply of tissues with oxygen (required in therapies with big losses of blood, such as surgeries or emergencies when donor blood is not available). Our own Institute is actively involved in solving these problems as having accumulated experience in studies and in synthesis of water-soluble synthetic and natural polymers. We also took part earlier in the development of plasma substitutes on the basis of polyvinil-pyrrolidon and polyvinyl alcohol. And it also looked like an attractive proposition using as a water-soluble polymer polyethyleneglycol which is harmless, binds large amounts of water, capable of destruction in the body and whose macromolecules have high mobility. But it was necessary to determine the conditions at which this substance will have the required molecular mass and will not have undesirable admixtures (catalysts, monomers or byproducts). Comprehensive experimental and clinical studies of a range of variants made it possible to optimize the composition and properties of the new plasma substitute and obtain a permission for its medical applications. It is called "Polyoxidine" and, as compared with such commonly used preparations as "Hemodez" and "Polyglucene", its polymer content has been reduced by 4 times. It also reduces blood viscosity, quickly restores blood supply in capillaries and prevent aggregation of formal elements of blood. As such it is used in cases of large losses of blood, post-traumatic and post-surgical shocks.
A different approach had to be adopted in the development of blood-substituting solutions which can not only restore the volume, electrolyte composition and osmotic pressure of plasma, but also perform the central gas-transport function of blood- reversible transport of oxygen from lungs into tissues. This function is performed in the body by a unique protein-hemoglobin which is present in high concentrations in "coreless" cells-erythrocytes, where it is protected from oxidation by special enzyme systems. And it should be noted at this point that the possibility of using hemoglobin solutions for sustaining animal life was demonstrated back in the 1930s. But hemoglobin as such possesses 5-times lower oxygen-transport potential as compared with erythrocytes, rapidly leaves the bloodstream (half-value period 1.5 - 2 h) and causes irreversible damage to kidneys. To overcome these problems our Institute researchers conducted studies in two directions.
The first, suggested by Professor Georgy Samsonov, consisted in modeling the gas- transport function of erythrocytes by way of sorptional immobilization of hemoglobin with specially synthesized hydrophilic polymer particles of micron size (diameter of erythrocyte-5 to 8 mem). As a result it was possible to produce polymer carriers capable of absorbing up to 15 g of protein per one kilo of its own mass. This considerably increased the effectiveness of oxygen release in com-
Comparison of "respiratory function" of erythrocytes, "artificial erythrocytes" and polyhemoglobin.
parison with hemoglobin solutions. And the obtained "cooperative" mechanism of oxygen binding is physiologically important because it permits the organism to have a rapid response to lower levels of the gas tissues by means of its release by hemoglobin in a narrow range of partial oxygen pressures between the lungs and body tissues. Biological tests of colloidal dispersions-"artificial erythrocytes"-demonstrated their high gas-transport potential (at the level of natural erythrocytes) and proved the theoretical possibility of their functioning of animal blood.
Another direction of our studies has been linked with the synthesis of high-molecular water-soluble derivatives of hemoglobin. In this case, for boosting oxygen-transport efficiency, we resorted to the polycondensation of the latter in combination with chemical "build-up" of synthetic regulator into its molecule. With the help of a special modifier, combining the function of an analog of the natural regulator of saturation of hemoglobin with oxygen with the properties of agent "suturing" hemoglobin molecules, we obtained polyhemoglobin which can transport O 2 just as well as erythrocytes. What is more, the chemical and intramolecular binding of protein molecules stabilizes the structure of hemoglobin. This eliminated its "natural" nephrotoxicity and prolongs functioning in the bloodstream (period of half- elimination 10 - 14h).
Broad pre-clinical studies of poly-hemoglobin solutions conducted at the Russian Scientific-Research Institute of Hematology and Trans-fusiology of the RF Ministry of Health demonstrated the high effectiveness and harmlessness of its solutions. In cases of losses of blood, practically incompatible with life (50 - 60 percent of the total volume) the rate of survival of animals reached 90 percent. This made it possible to recommend polyhemoglobin solutions as the basic component of the blood-substitute preparation "Gelenpol" which can be administered to any patient without preliminary compatibility tests. And the preparation retains its properties for two years. During clinical tests its was administered in dozes of up to 2 litres to surgical patients with heavy blood losses and also in cases of post-traumatic shock of II and III degrees. The preparation invariably demonstrated its high efficiency: improved blood supply and oxygen regime of organism, normalized arterial pressure and stimulated hemopoiesis. "Gelenpol" has been cleared for clinical use and registered by Russian Ministry of Health.
Summing it up, the polymer trend in drug therapy, put forward more than 50 years ago by our outstanding scholar S. Ushakov, has proved to be a very fruitful one. Supported by fellow-scientists and developed by his pupils, it has turned into a branchy tree producing healing fruits in the form of new original medicinal preparations for the benefit of thousands of patients.
The RF State Prize for Science and Technology for 2001 has been awarded to academicians Rem Petrov and Viktor Kabanov, Academician of the Russian Academy of Medical Sciences Rakhim Khaitov, Arkadiy Nekrasov, Dr. Sc. (Chem.) and Ravshan Ataullakhanov, Dr. Sc. (Med.), for the development and introduction into Russian medical practice of conjugated polymer-subunit immunogens and vaccines. These preparations were obtained through chemical binding (conjugation) of antigens with immunostimulating synthetic polyelectrolyte. They are harmless, prone to destruction and are easily evacuated from the body of a patient. Moscow scientists have developed on this basis the "Grippol" vaccine which contains purified proteins-hemagglutinin and neurominidaze of three viruses of influenza A and B. Its commercial production was started in 1997 by "Immunopreparat" State Unitary Enterprise. Vaccinated from 1997 to 2002 have been more than 35 mln children and adults, demonstrating high disease-preventive potential on the level of the most advanced imported analogues at a much lower antigen load and much lower prices.
Illustrations provided by the authors.
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