Disease modifying drugs. Basic antirheumatic drugs: the view of a clinical pharmacologist. Common side effects of disease-modifying antirheumatic drugs

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An excerpt characterizing disease-modifying antirheumatic drugs

Having taken a fancy to the tiny Montsegur, which was the most magical castle in the Valley (as it stood at the “transition point” to other worlds), Magdalena and her daughter soon began to slowly move there. They began to settle in their new, still unfamiliar, House ...
And, finally, remembering Radomir's persistent desire, Magdalena gradually began to recruit her first students ... This was probably one of the easiest tasks, since every person on this marvelous piece of land was more or less gifted. And almost everyone was hungry for knowledge. Therefore, very soon Magdalene already had several hundred very diligent students. Then this figure grew into a thousand... And very soon the entire Valley of Magicians was covered by her teachings. And she took as many people as possible to divert herself from her bitter thoughts, and she was inexpressibly glad at how greedily the Occitans were drawn to Knowledge! She knew that Radomir would be glad of this from the bottom of his heart... and recruited even more applicants.
- Sorry, Sever, but how did the Magi agree with this ?!. After all, they so carefully protect their Knowledge from everyone? How did the Lord let this happen? Did Magdalene teach everyone, not choosing only the initiates?
– Vladyka never agreed with this, Isidora... Magdalena and Radomir went against his will, revealing this knowledge to people. And I still don't know which one of them was really right...

Genetic engineering and drugs

Before the advent of recombinant DNA technology, many drugs based on human proteins could only be obtained in small quantities, their production was very expensive, and the mechanism of biological action was sometimes poorly understood. With the help of the new technology, the whole range of such drugs is obtained in quantities sufficient both for their effective testing and for use in the clinic. To date, more than 400 genes have been cloned (mostly in the form of cDNA) of various human proteins that can become drugs. Most of these genes are already expressed in host cells, and their products are now used to treat various human diseases. As usual, they are first tested on animals, and then rigorous clinical trials are carried out. The annual volume of the world market of drugs based on human proteins is about 150 billion dollars and is constantly growing. The volume of the world market for medicines based on recombinant proteins is increasing by 12-14% per year and in 2000 amounted to about 20 billion dollars.

On the other hand, the use of specific antibodies as therapeutic agents is promising. They are used to neutralize toxins, fight bacteria, viruses, and treat cancer. The antibody either neutralizes the "intruder" - a foreign agent, or destroys a specific target cell. Despite their promising potential, antibodies have so far been rarely used to prevent or treat disease. And only with the development of recombinant DNA technology and the development of methods for obtaining monoclonal antibodies and with the decoding of the molecular structure and function of immunoglobulins, commercial interest in the use of specific antibodies for the treatment of various diseases again arose.

The development of new methods for the prevention and treatment of many human diseases has made a huge contribution to the growth of people's well-being in the 20th century. However, this process cannot be considered complete. The so-called "old" diseases, such as malaria, tuberculosis, etc., may reappear as soon as preventive measures are weakened, or resistant strains appear. A typical situation in this respect is in Ukraine and Russia.

Antibiotics are low molecular weight substances that differ in chemical structure. What these compounds have in common is that, being products of vital activity of microorganisms, they specifically disrupt the growth of other microorganisms in negligible concentrations.

Most antibiotics are secondary metabolites. They, like toxins and alkaloids, cannot be classified as substances strictly necessary to ensure the growth and development of microorganisms. On this basis, secondary metabolites differ from the primary ones, in the presence of which the death of the microorganism occurs.

The biosynthesis of antibiotics, as well as other secondary metabolites, usually occurs in cells that have stopped growing (idiophase). Their biological role in ensuring the vital activity of producer cells remains unexplored to the end. Experts studying the prospects of biotechnology in the field of microbiological production of antibiotics believe that under unfavorable conditions, they suppress the growth of competing microorganisms, thereby providing more favorable conditions for the survival of the microbe-producer of one or another antibiotic. The significance of the process of antibiotic formation in the life of a microbial cell is confirmed by the fact that in streptomycetes, about 1% of genomic DNA falls on the share of genes encoding enzymes for the biosynthesis of antibiotics, which may not be expressed for a long time. The producers of known antibiotics are mainly six genera of filamentous fungi, three genera of actinomycetes (almost 4000 different antibiotics) and two genera of true bacteria (about 500 antibiotics). Of the filamentous fungi, special attention should be paid to mold fungi of the genera Cephalosporium and Penicillium, which are producers of the so-called beta-lactam antibiotics - penicillins and cephalosporins. Most actinomycetes synthesizing antibiotic substances, including tetracyclines, belong to the genus Streptomyces.

Of the known 5000-6000 natural antibiotic substances, only about 1000 are produced for sale to consumers. At the time when the antibacterial effect of penicillin and the possibility of its use as a drug were established (H.W. Flory, E.B. Chain et al., 1941 ), the productivity of the laboratory mold strain - 2 mg of the preparation per 1 liter of culture liquid - was clearly insufficient for the industrial production of the antibiotic. By repeated systematic exposure of the original strain of Penicillium chrisogenum to such mutagens as X-ray and ultraviolet irradiation, nitrogen mustard, in combination with spontaneous mutations and selection of the best producers, it was possible to increase the productivity of the fungus by 10,000 times and bring the concentration of penicillin in the culture liquid to 2%.

The way to increase the efficiency of antibiotic-producing strains, based on random mutations and which has become classic, despite the enormous labor costs, is still used. This situation is a consequence of the fact that an antibiotic, unlike a protein, is not the product of a particular gene; antibiotic biosynthesis occurs as a result of the joint action of 10-30 different enzymes encoded by the corresponding number of different genes. In addition, for many antibiotics, the microbiological production of which has been established, the molecular mechanisms of their biosynthesis have not yet been studied. The polygenic mechanism underlying the biosynthesis of antibiotics is the reason that changes in individual genes are not successful. Automation of routine techniques for analyzing the productivity of mutants makes it possible to study tens of thousands of functioning strains and thereby speeds up the selection procedure when using the classical genetic technique.

A new biotechnology based on the use of strains-superproducers of antibiotics implies the improvement of the mechanisms of protection of the producer from the antibiotic synthesized by him.

High productivity is shown by strains that are resistant to high concentrations of antibiotics in the culture medium. This property is also taken into account when designing superproducer cells. Since the discovery of penicillin in the late 1920s, more than 6,000 antibiotics have been isolated from various microorganisms, with different specificities and different mechanisms of action. Their widespread use in the treatment of infectious diseases has helped save millions of lives. The vast majority of the major antibiotics have been isolated from the Gram-positive soil bacterium Streptomyces, although fungi and other Gram-positive and Gram-negative bacteria also produce them. 100,000 tons of antibiotics are produced annually worldwide, valued at about S billion dollars, of which more than $100 million is accounted for by antibiotics added to livestock feed as additives or growth promoters.

It is estimated that between 100 and 200 new antibiotics are discovered every year by scientists, primarily as part of extensive research programs to find among thousands of different microorganisms those that would synthesize unique antibiotics. Obtaining and clinical trials of new drugs are very expensive, and only those that have great therapeutic value and are of economic interest go on sale. They account for 1-2% of all detected antibiotics. Recombinant DNA technology has a great effect here. First, it can be used to create new antibiotics with a unique structure that have a more powerful effect on certain microorganisms and have minimal side effects. Secondly, genetic engineering approaches can be used to increase the yield of antibiotics and, accordingly, to reduce the cost of their production.

It can be considered that clinical biotechnology originated with the beginning of the industrial production of penicillin in the 1940s. and its use in therapy. Apparently, the use of this first natural penicillin contributed to the reduction of morbidity and mortality more than any other drug, but, on the other hand, posed a number of new problems that were again solved with the help of biotechnology.

Firstly, the successful use of penicillin caused a great demand for this drug, and to meet it, it was necessary to dramatically increase the yield of penicillin in its production. Secondly, the first penicillin - C (benzylpenicillin) - acted mainly on gram-positive bacteria (for example, Streptococci and Staphylococci), and it was necessary to obtain antibiotics with a broader spectrum of action and / or activity, affecting and gram-negative bacteria such as E. coli and Pseudomonas. Thirdly, since antibiotics caused allergic reactions (most often minor, like a skin rash, but sometimes more severe, life-threatening manifestations of anaphylaxis), it was necessary to have a whole set of antibacterial agents in order to be able to choose from among equally effective drugs one that would not cause an allergic reaction in the patient. Fourth, penicillin is unstable in the acidic environment of the stomach and should not be given orally. Finally, many bacteria are becoming resistant to antibiotics. A classic example of this is the formation of the enzyme penicillinase (more correctly, beta-lactamase) by staphylococci, which hydrolyzes the amide bond in the beta-lactam ring of penicillin to form pharmacologically inactive penicilloic acid. It was possible to increase the yield of penicillin during its production mainly due to the consistent use of a series of mutants of the original strain Penicillium chrysogenum, as well as by changing the growing conditions.

The process of biosynthesis of one antibiotic can consist of dozens of enzymatic reactions, so cloning all the genes for its biosynthesis is not an easy task. One approach to isolating a complete set of such genes is based on the transformation of one or several mutant strains that are not capable of synthesizing a given antibiotic with a clone bank created from the chromosomal DNA of a wild-type strain. After the introduction of the bank of clones into mutant cells, the selection of transformants capable of synthesizing the antibiotic is carried out. Then, plasmid DNA of a clone containing a functionally expressed antibiotic gene (i.e., a gene that restores the function lost by the mutant strain) is isolated and used as a probe for screening another clone bank of chromosomal DNA of the wild-type strain, from which clones containing nucleotide sequences that overlap with the probe sequence. In this way, the DNA elements adjacent to the complementing sequence are identified and then cloned and the complete antibiotic biosynthesis gene cluster is recreated. The described procedure refers to the case when these genes are grouped in one site of chromosomal DNA. If, on the other hand, the biosynthesis genes are scattered in small clusters at different sites, then at least one mutant per cluster is needed to obtain DNA clones that can be used to identify the rest of the cluster genes.

Using genetic or biochemical experiments, it is possible to identify and then isolate one or more key biosynthetic enzymes, determine their N-terminal amino acid sequences, and, based on these data, synthesize oligonucleotide probes. This approach was used to isolate the isopenicillin N synthetase gene from Penicillium chrysogenum. This enzyme catalyzes the oxidative condensation of 5-(1_-a-aminoadipylN-cysteinyl-P-valine) to isopenicillin N, a key intermediate in the biosynthesis of penicillins, cephalosporins, and cephamycins.

New antibiotics with unique properties and specificity can be obtained by genetic engineering manipulations with genes involved in the biosynthesis of already known antibiotics. One of the first experiments in which a new antibiotic was obtained was to combine in one microorganism two slightly different pathways for the biosynthesis of an antibiotic.

One of the Streptomyces plasmids, plJ2303, carrying a 32.5 kb fragment of the chromosomal DNA of S. coelicoior, contains all the genes of the enzymes responsible for the biosynthesis of the antibiotic actinorhodin, a member of the isochromanquinone antibiotic family, from acetate. The whole plasmid and various subclones carrying portions of the 32.5 kb fragment (e.g., plJ2315) were introduced into either the AM-7161 strain of Streptomyces sp.T, which synthesizes the related antibiotic medermicin, or into the B1140 or Tu22 strain of S. violaceoruber synthesizing related antibiotics granaticin and dihydrogranaticin.

All of these antibiotics are acid-base indicators that give the growing culture a characteristic color that depends on the pH of the medium. In turn, the pH (and color) of the medium depends on which compound is being synthesized. Mutants of the parent strain S.coelicoior, unable to synthesize actinorodin, are colorless. The appearance of color after the transformation of strain AM-7161 Streptomyces sp. or strains B1J40 or Tu22 S.violaceoruber with a plasmid carrying all or several genes encoding actinorhodin biosynthesis enzymes indicates the synthesis of a new antibiotic Transformants of strain AM-7161 Streptomyces sp. and strain-6 1140 S.violaceoruber, containing the plasmid pM2303, synthesize antibiotics encoded by both the plasmid and chromosomal DNA.

However, when the Tu22 strain of S.violaceoruber is transformed with the plJ2303 plasmid, a new antibiotic, dihydrogranatirodine, is synthesized along with actinorhodin, and when the AM-7161 strain of Streptomyces sp. Another new antibiotic, mederrhodin A, is synthesized by the plJ2315 plasmid.

Structurally, these new antibiotics differ little from actinorhodin, medermycin, granaticin, and hydrogranaticin and are likely formed when an intermediate product of one biosynthetic pathway serves as a substrate for an enzyme in another pathway. When the biochemical properties of various pathways of antibiotic biosynthesis are studied in detail, it will be possible to create new unique highly specific antibiotics by manipulating the genes that encode the corresponding enzymes.

The term "polyketide" refers to a class of antibiotics that are formed by the sequential enzymatic condensation of carboxylic acids such as acetate, propionate and butyrate. Some polyketide antibiotics are synthesized by plants and fungi, but most of them are produced by actinomycetes as secondary metabolites. Before manipulating the genes encoding enzymes for the biosynthesis of polyketide antibiotics, it was necessary to elucidate the mechanism of action of these enzymes.

Having studied in detail the genetic and biochemical components of erythromycin biosynthesis in Saccharopolyspora erythraea cells, it was possible to make specific changes in the genes associated with the biosynthesis of this antibiotic and to synthesize erythromycin derivatives with other properties. First, the primary structure of the S.erythraea DNA fragment was determined! 56 kb containing the ery gene cluster, then modified with erythromycin polyketide synthase in two different ways. To do this, 1) the DNA region encoding beta-ketoreductase was removed, or 2) a change was made to the DNA region encoding enoyl reductase. These experiments made it possible to show experimentally that if a cluster of genes encoding enzymes for the biosynthesis of a certain polyketide antibiotic is identified and characterized, then, by making specific changes to them, it will be possible to change the structure of the antibiotic in a targeted manner.

In addition, by cutting and joining certain sections of DNA, it is possible to move the polyketide synthase domains and obtain new polyketide antibiotics.

With the help of genetic engineering, it is possible not only to create new antibiotics, but also to increase the efficiency of the synthesis of already known ones. The limiting factor in the industrial production of antibiotics using Streptomyces spp. often is the amount of oxygen available to the cells. Due to the poor solubility of oxygen in water and the high density of the culture of Streptomyces, it is often insufficient, cell growth slows down, and the yield of the antibiotic decreases. To solve this problem, it is possible, firstly, to change the design of the bioreactors in which the culture of Streptomyces is grown, and secondly, using genetic engineering methods, to create strains of Streptomyces that use the available oxygen more efficiently. These two approaches are not mutually exclusive.

One of the strategies used by some aerobic microorganisms to survive in conditions of lack of oxygen is the synthesis of a hemoglobin-like product that can store oxygen and deliver it to cells. For example, the aerobic bacterium Vitreoscilla sp. synthesizes a homodimeric heme-containing protein, functionally similar to eukaryotic hemoglobin. The Vitreoscilla "hemoglobin" gene was isolated, inserted into the Streptomyces plasmid vector, and introduced into the cells of this microorganism. After its expression, Vitreoscilla hemoglobin accounted for approximately 0.1% of all S. coelicoior cellular proteins, even when expression was under the control of Vitreoscilla's own hemoglobin gene promoter, and not the Streptomyces promoter. Transformed S.coelicoior cells growing at a low content of dissolved oxygen (approximately 5% of the saturating concentration) synthesized 10 times more actinorhodin per 1 g of dry cell mass and had a higher growth rate than nontransformed ones. This approach can also be used to provide oxygen to other microorganisms growing in oxygen-deficient conditions.

The starting material for the chemical synthesis of some cephalosporins - antibiotics with minor side effects and active against many bacteria - is 7-aminocephalosporanic acid (7ACA), which in turn is synthesized from the antibiotic cephalosporin C. Unfortunately, natural microorganisms capable of synthesizing 7ACA , has not yet been identified.

A novel 7ACA biosynthetic pathway was constructed by incorporating specific genes into the plasmid of the fungus Acremonium chrysogenum, which normally only synthesizes cephalosporin-C. One of these genes was from Fusarium solani cDNA encoding D-amino acid oxidase, while the other was derived from Pseudomonas diminuta genomic DNA and encoded cephalosporin acylase. In the plasmid, the genes were under the control of the A. chrysogenum promoter. In the first step of the new biosynthetic pathway, cephalosporin-C is converted to 7-p-(5-carboxy-5-oxopentanamide) cephalosporan acid (keto-AO-7ACA) by amino acid oxidase. A portion of this product reacts with hydrogen peroxide, one of the by-products, to form 7-beta-(4-carboxybutanamide)-cephalosporan acid (GL-7ACA). Both cephalosporin-C, keto-A0-7ACA and GL-7ACA can be hydrolyzed by cephalosporin acylase to form 7ACA, however only 5% of cephalosporin-C is directly hydrolyzed to 7ACA. Therefore, both enzymes are required for the formation of 7ACA in high yield.

In the late 70s - early 80s. XX century DNA technology for the first time began to attract the attention of the public and large investors. One of the promising biotechnological products was interferon, which at the time was hoped as a miracle cure against a variety of viral diseases and cancer. The isolation of human interferon cDNA and its subsequent expression in Escherichia coll was reported by all interested publications in the world.

Different approaches are used to isolate human genes or proteins. Usually, the desired protein is isolated and the amino acid sequence of the corresponding region of the molecule is determined. Based on this, the nucleotide sequence encoding it is found, the corresponding oligonucleotide is synthesized, and it is used as a hybridization probe to isolate the desired gene or cDNA from genomic or cDNA libraries. Another approach is to generate antibodies to the purified protein and use them to screen libraries that express certain genes. For human proteins synthesized predominantly in a single tissue, a cDNA library derived from mRNA isolated from that tissue will be enriched in the target DNA sequence. For example, the main protein synthesized by the cells of the islets of Langerhans of the pancreas is insulin, and 70% of the mRNA isolated from these cells encode it.

However, the principle of cDNA enrichment is inapplicable for those human proteins, the amount of which is very small or the place of synthesis of which is unknown. In this case, other experimental approaches may be needed. For example, human interferons (IF), including alpha, beta and gamma interferons, are natural proteins, each of which can find its own therapeutic application. The first interferon gene was isolated in the early 1980s. XX century. Since then, several different interferons have been discovered. A polypeptide that has the effect of human leukocyte interferon is synthesized in E. coli.

Several features of interferon have made the isolation of its cDNA particularly difficult. First, despite the fact that interferon was purified by more than 80,000 times, it was possible to obtain it only in very small quantities, because. its exact molecular weight was not known at the time. Secondly, unlike many other proteins, interferon does not have an easily identifiable chemical or biological activity: it was evaluated only by reducing the cytopathic effect of an animal virus on cell culture, and this is a complex and lengthy process. Thirdly, unlike insulin, it was not known whether there are human cells capable of producing interferon in sufficiently large quantities, i.e. whether there is a source of interferon mRNA. Despite all these difficulties, the cDNA encoding interferon was eventually isolated and characterized. When isolating their cDNA, a special approach had to be developed to overcome the difficulties associated with the insufficient content of the corresponding mRNAs and proteins. Now such a procedure for DNA extraction is common and standard, and for interferons it is as follows.

1. mRNA was isolated from human leukocytes and fractionated by size; reverse transcription was performed and inserted into the Psti site of plasmid pBR322.

2. The resulting product was transformed into Escherichia coli. The resulting clones were divided into groups. Testing was carried out on a group of clones, which made it possible to speed up the process of their identification.

3. Each group of clones was hybridized with a crude preparation of IF-mRNA.

4. From the resulting hybrids containing cloned DNA and mRNA, mRNA was isolated and translated in a cell-free protein synthesis system.

5. Determined the interferoic antiviral activity of each mixture obtained as a result of translation. Groups that showed interferon activity contained a clone with cDNA hybridized with IF-mRNA.

6. Positive groups were split into subgroups containing multiple clones and tested again. Subgrouping was repeated until a clone containing full-length human IF-cDNA was identified.

Since then, several different types of interferons have been discovered. The genes of several interferons were isolated and their effectiveness in the treatment of various viral diseases was shown, but, unfortunately, interferon did not become a panacea.

Based on the chemical and biological properties of interferon, three groups can be distinguished: IF-alpha, IF-beta and IF-gamma. IF-alpha and IF-beta are synthesized by cells treated with virus or viral RNA preparations, and IF-gamma is produced in response to substances that stimulate cell growth. IF-alpha is encoded by a gene family that includes at least 15 non-allelic genes, while IF-beta and IF-gamma are encoded by one gene each. Subtypes of IF-alpha exhibit different specificities. For example, when testing the effectiveness of IF-elfa-1 and IF-alpha-2 on a virus-treated bovine cell line, these interferons exhibit similar antiviral activity, while in the case of virus-treated human cells, IF-alpha-2 is seven times more active than IF- alpha 1. If antiviral activity is tested on mouse cells, then IF-alpha-2 is 30 times less effective than IF-alpha-1.

Due to the fact that there is a family of interferons, several attempts have been made to create IF with combined properties, using the fact that different members of the IF-alpha family differ in the degree and specificity of their antiviral activity. Theoretically, this can be achieved by combining parts of the gene sequences of different IF-alpha. This will result in a fusion protein with different properties than either of the original proteins. Comparison of the IF-alpha-1 and IF-alpha-2 cDNA sequences showed that they contain the same restriction sites at positions 60, 92, and 150. After cleavage of both cDNA at these sites and subsequent ligation of the fragments, several hybrid genes were obtained. These genes were expressed in E. coli, the synthesized proteins were purified and their biological functions were examined. Testing of the protective properties of hybrid IFs in mammalian cell culture showed that some of them are more active than parental molecules. In addition, many hybrid IFs induced the formation of 2'-5'-oligoisoadenylate synthetase in control cells. This enzyme is involved in the synthesis of 2'-5'-linked oligonucleotides, which in turn activate the latent cellular endoribonuclease, which cleaves viral mRNA. Other hybrid IFs showed greater antiproliferative activity than the parent molecules in cultures of various human cancer cells.

The strategy of constructing new proteins by replacing functional domains or by site-directed mutagenesis can be used to increase or decrease the biological property of a protein. For example, native human growth hormone (hGH) binds to both the growth hormone receptor and the prolactin receptor in different cell types. To avoid unwanted side effects during treatment, it is necessary to exclude the attachment of hGH to the prolactin receptor. Since the portion of the growth hormone molecule that binds to this receptor only partially coincides in its amino acid sequence with the portion of the molecule that interacts with the prolactin receptor, it was possible to selectively reduce the binding of the hormone to the latter. For this, site-specific mutagenesis was used, as a result of which certain changes occurred in the side groups of some amino acids (His-18, His-21 and Glu-174) - ligands for Zn 2+ ions necessary for high-affinity binding of hGH to the prolactin receptor. The modified growth hormone binds only to its "own" receptor. The results obtained are of undoubted interest, but it is still unclear whether the modified hGH will be able to find application in the clinic.

The most common lethal hereditary disease among Caucasians is cystic fibrosis. There are 30,000 cases of this disease in the USA, 23,000 in Canada and Europe. Patients with cystic fibrosis often suffer from infectious diseases that affect the lungs. Treatment of recurrent infections with antibiotics eventually leads to the emergence of resistant strains of pathogenic bacteria. Bacteria and their lysis products cause the accumulation of viscous mucus in the lungs, making it difficult to breathe. One of the components of mucus is high molecular weight DNA, which is released from bacterial cells during lysis. Scientists from the biotechnology company Genentech (USA) have isolated and expressed the gene for DNase, an enzyme that breaks down high-molecular DNA into shorter fragments. The purified enzyme is injected as part of an aerosol into the lungs of patients with cystic fibrosis, it cleaves DNA, the viscosity of the mucus decreases, which makes it easier to breathe. Although these measures do not cure cystic fibrosis, they alleviate the condition of the patient. This enzyme was recently approved by the US Food and Drug Administration and sold approximately $100 million in 2000.

Another biotechnological product that helps patients is alginate lyase. Alginate is a polysaccharide synthesized by a variety of algae, as well as soil and marine bacteria. Its monomeric units are two saccharides - beta-D-mannuronate and alpha-1-guluronate, the relative content and distribution of which determine the properties of a particular alginate. Thus, a-L-guluronate residues form interchain and intrachain crosslinks by binding calcium ions; beta-D-mannuronate residues bind other metal ions. Alginate containing such crosslinks forms an elastic gel whose viscosity is directly proportional to the size of the polysaccharide molecules.

The release of alginate by mucous strains of Pseudomonas aeruginosa significantly increases the viscosity of mucus in patients with cystic fibrosis. To clear the respiratory tract and alleviate the condition of patients, in addition to treatment with DNase, depolymerization of alginate using alginate lyase should be carried out.

The alginate lyase gene was isolated from Flavobacterium sp., a Gram-negative soil bacterium that actively produces this enzyme. Based on E. coli, a bank of Flavobacterium clones was created and those that synthesize alginate lyase were screened by seeding all clones on a solid medium containing alginate with the addition of calcium ions. Under such conditions, all alginate in the medium, except for that which surrounds the alginate-lyase-producing colonies, forms crosslinks and becomes cloudy. Hydrolyzed alginate loses its ability to form crosslinks, so the environment around the alginate-lyase-synthesizing colonies remains transparent. Analysis of a cloned DNA fragment present in one of the positive colonies showed the presence of an open reading frame encoding a polypeptide with a molecular weight of about 69,000. Flavobacterium sp. First, some proteolytic enzyme cuts off an N-terminal peptide with a mass of about 6000 from it. The remaining protein with a molecular weight of 63,000 is able to depolymerize the alginate produced by both bacteria and algae. When it is subsequently cut, a product with a molecular weight of 23,000 is formed, which depolymerizes seaweed alginate, and an enzyme with a molecular weight of 40,000, which destroys bacterial alginate. To obtain large amounts of the enzyme with a molecular weight of 40,000, the DNA encoding it was amplified by polymerase chain reaction (PCR) and then inserted into a plasmid vector isolated from B.subrjlis, carrying the gene encoding the B.subrjlis α-amylase signal peptide. Transcription was controlled using a penicillinase gene expression system. When B. subrjlis cells were transformed with the obtained plasmid and seeded on a solid medium containing alginate with the addition of calcium ions, colonies with a large halo were formed. When such colonies were grown in liquid medium, the recombinant alginate lyase was released into the culture medium. Subsequent tests showed that this enzyme was able to effectively liquefy the alginates produced by the mucilaginous strains of P. aeruginosa that were isolated from the lungs of patients with cystic fibrosis. More research is needed to determine whether clinical testing of recombinant alginate lyase is appropriate.

In the 1970s views on passive immunization were revised: it began to be considered a preventive means of combating the rejection of transplanted organs. It was proposed to introduce patients with specific antibodies that would bind to certain types of lymphocytes, reducing the immune response directed against the transplanted organ.

The first substances recommended by the US Food and Drug Administration for use as immunosuppressants in human organ transplants were murine monoclonal antibodies OCTH. The so-called T-cells are responsible for the rejection of organs - lymphocytes that differentiate in the thymus. OCTZ binds to a receptor found on the surface of any T cell called CD3. This prevents the development of a complete immune response and rejection of the transplanted organ. This immunosuppression is very effective, although it has some side effects, such as fever and rashes.

Techniques have been developed for the production of antibodies using E. coli. Hybridomas, like most other animal cell cultures, grow relatively slowly, do not reach high densities, and require complex and expensive media. Monoclonal antibodies obtained in this way are very expensive, which does not allow them to be widely used in the clinic.

To solve this problem, attempts have been made to create a kind of "bioreactors" based on genetically modified bacteria, plants and animals. For this purpose, gene constructs capable of encoding individual antibody regions were introduced into the host genome. For effective delivery and functioning of some immunotherapeutic agents, one antigen-binding region of an antibody (Fab or Fv fragment) is often sufficient; the presence of an Fc fragment of an antibody is optional.

Today, the prospects of agricultural biotechnology to provide such plants that will be used as medicines or vaccines look more and more real. It is hard to imagine how important this could be for poor countries, where conventional pharmaceuticals are still a novelty and traditional WHO vaccination programs are proving too expensive and difficult to implement. This line of research should be supported in every possible way, including through cooperation between the public and private sectors of the economy.

Among the genes whose expression in plants is considered exotic, the most important are those encoding the synthesis of polypeptides of medical importance. Obviously, the Calgene patent on the expression of mouse interferon in plant cells should be considered the first study in this area. Later, the synthesis of immunoglobulins in plant leaves was shown.

In addition, it is possible to introduce into the plant genome a gene encoding the envelope protein (proteins) of a virus. By consuming the plant as food, people will gradually acquire immunity to this virus. In fact, this is the creation of plant medicines.

Transgenic plants have a number of advantages over microbial, animal, and human cell culture for the production of recombinant proteins. Among the advantages of transgenic plants, we note the main ones: the possibility of large-scale production, low cost, ease of purification, the absence of impurities that have allergenic, immunosuppressive, carcinogenic, teratogenic and other effects on humans. Plants can synthesize, glycosylate, and assemble mammalian proteins from subunits. When eating raw vegetables and fruits that carry genes encoding the synthesis of protein vaccines, oral immunization occurs.

One way to reduce the risk of gene leakage into the environment, which is used, in particular, in the creation of edible vaccines, is to introduce foreign genes into chloroplasts, and not into nuclear chromosomes, as usual. It is believed that this method will expand the scope of GM plants. Despite the fact that it is much more difficult to introduce the desired genes into chloroplasts, this method has several advantages. One of them is that foreign DNA from chloroplasts cannot get into the pollen. This completely eliminates the possibility of uncontrolled transfer of GM material.

A promising direction is the creation of transgenic plants carrying genes for proteins characteristic of bacteria and viruses that cause infectious diseases. When raw fruits and vegetables carrying such genes or their sublimated juices are consumed, the body is vaccinated. For example, when introducing the gene for the non-toxic cholera enterotoxin subunit into potato plants and feeding raw tubers to experimental mice, antibodies to cholera pathogens were formed in their bodies. It is clear that such edible vaccines can be an effective, simple and inexpensive way to protect people and ensure food safety in general.

The development of DNA technology in recent decades has also revolutionized the development and production of new vaccines. Using the methods of molecular biology and genetic engineering, the antigenic determinants of many infectious agents have been identified, the genes encoding the corresponding proteins have been cloned, and, in some cases, the production of vaccines based on the protein subunits of these antigens has been established. Diarrhea caused by infection with Vibrio cholerae or enterotoxigenic Escherichia coli (Escherichia coli) is one of the most dangerous diseases with a high percentage of deaths, especially in children. The total number of cases of cholera on the globe exceeds 5 million cases annually, resulting in the death of about 200 thousand people. Therefore, the World Health Organization (WHO) pays attention to the prevention of diarrheal infections, in every possible way stimulating the creation of a variety of vaccines against these diseases. Outbreaks of cholera are also found in our country, especially in the southern regions.

Diarrheal bacterial diseases are also widespread in farm animals and poultry, especially in young animals, which is the cause of large losses in farms as a result of weight loss and mortality.

The classic example of a microbial recombinant vaccine is the production of hepatitis B surface antigen. The HBsAg viral gene has been inserted into a yeast plasmid, resulting in the production of large quantities of the viral protein in yeast, which, after purification, is used for injection as an effective vaccine against hepatitis (Pelre et al., 1992).

Many southern countries with a high incidence of hepatitis carry out universal vaccination of the population, including children, against this disease. Unfortunately, the cost of such a vaccine is relatively high, which prevents widespread universal vaccination programs in countries with a low standard of living. In connection with this situation, in the early 1990s, WHO took the initiative to create new technologies for the production of inexpensive vaccines against infectious diseases, accessible to all countries of the world.

Ten years ago, the concept of using transgenic plants for the production of so-called "edible" vaccines was put forward. Indeed, if any edible plant organ synthesizes an antigen protein with strong oral immunogenic properties, then when these plants are eaten, the antigen protein will be absorbed in parallel with the production of appropriate antibodies.

Tobacco plants carrying the gene encoding the hepatitis B envelope antigen under the plant promoter were obtained. The presence of the antigen in the leaves of transgenic plants was confirmed by enzyme immunoassay. The similarity of the physicochemical structure and immunological properties of the resulting recombinant antigen and human serum antigen is shown.

The identification of antibodies produced in plants showed the possibility of assembling two recombinant gene products into one protein molecule, which is impossible in prokaryotic cells. Assembly of antibodies occurred when both chains were synthesized with a signal sequence. In this case, along with the possibility of introducing two genes into one plant, it is also possible to combine individual polypeptide chains synthesized in different transgenic plants into a complete protein during hybridization of these two plants. It is possible to introduce several genes on one plasmid.

Transgenic plants producing autoantigens can also be used in other autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, insulin-dependent diabetes, and even organ transplant rejection. Insulin-dependent diabetes is an autoimmune disease in which the insulin-producing cells of the pancreas are destroyed by their own cytotoxic T-lymphocytes. Oral prophylactic intake of significant amounts of immunogenic proteins can lead to prevention and a significant delay in the onset of symptoms of autoimmune diseases. However, it is possible only in the presence of a significant amount of autoantigens. The proteins insulin and pancreatic glutamic acid decarboxylase (GAD65) are considered as oral vaccines to prevent insulin dependent diabetes. Recently, Canadian biotechnologists have obtained transgenic potato plants that synthesize pancreatic glutamic acid decarboxylase. When fed to diabetic mice, both the incidence of diabetes and the magnitude of the autoimmune response were reduced.

The above results of genetic engineering developments convincingly indicate the possibility of creating "edible" vaccines based on transgenic plants. Given the fact that the development of vaccines for humans will require much more time and more thorough testing for harm to health, it should be expected that the first edible vaccines will be developed for animals. Animal studies will help uncover the mechanisms of action of "edible" vaccines, and only then, after a long study and comprehensive evaluation, such vaccines can be used in clinical practice. Nevertheless, work in this direction is actively continuing, and the idea of ​​using plants for the production of vaccines has already been patented in the United States, which indicates a commercial interest in these developments.

Despite these encouraging results, the problem of creating commercial "edible" vaccines against diarrhea requires further research. In the pathogenesis of the enterotoxic form of bacterial and cholera diarrhea, the primary task is to enable bacteria to multiply in the small intestine. This process depends on the ability of Escherichia coli to adhere, which is due to the presence on the surface of bacterial cells of special filamentous formations of a protein nature - pili. Significantly more bacteria are found on the walls of the small intestine of patients with diarrhea than in the lumen of the same part of the intestine, which is associated with the presence of fimbrial adhesins in Escherichia coli - proteins that provide binding to receptors on the surface of the intestinal epithelium.

Even non-pathogenic strains of Escherichia coll, which contained a plasmid encoding adhesin synthesis, were able to colonize the intestine and cause diarrhea without producing enterotoxins. In this regard, it is likely that immunity against toxins alone will not be sufficient to prevent pathogenic effects caused by V. cholerae or E. coli. It is possible that to overcome these effects, in addition to the enterotoxin antigens, it will be necessary to express neutralizing epitopes of structural antigens such as lipopolysaccharides, bacterial outer membrane proteins, or pili-associated adhesins of these bacteria responsible for binding to the intestinal mucosa. Recently, one such adhesin, FimH, has been successfully used to immunize mice against bacterial diarrhea.

Another important problem associated with the development of "edible" vaccines is the level of expression of a heterologous antigen in plants. Because oral vaccines require larger amounts of antigen than parenteral vaccines, the amount of antigen synthesized in plants, which currently amounts to no more than 0.3% of the total soluble protein, must be increased. At the same time, the level of expression must be high enough to induce an immune response, but be less than the level that induces tolerance to the antigen, as is the case with substances consumed with ordinary food. And since the immune response (immunogenicity versus tolerance) can be antigen-specific, the expression levels for each potential antigen will need to be selected individually.

As experiments show, the level of expression of a heterologous antigen in plants can be increased by using tissue-specific promoters and enhancers, enhancers of transcription and translation, by adding transport peptides, as well as by changing the nucleotide sequence of the corresponding genes using plant-preferred codons. However, the question of which plants are better to use and in which edible organ it is better to express the antigen requires further research, since various plants may contain substances that block or slow down the immune response or are simply toxic to humans and animals, such as alkaloids. in tobacco cells.

Achievements of scientific and technological progress have affected all spheres of human activity, from production to everyday life. For centuries, people have sought to free themselves from physical exertion by automating production, creating household appliances, and so on. And, in general, they were released. As a result, by the end of the 20th century, the daily energy consumption of a person decreased by 1.5-2 times compared to its beginning.

Human health is determined mainly by hereditary predisposition (genetics) and nutrition. At all times, the creation of a food base has been the key and basis for the prosperity of any state. Therefore, any state is interested in prevention projects and health programs, improving the structure of nutrition, improving the quality of life, reducing morbidity and mortality. It is nutrition that closely connects us with the environment, and food is the material from which the human body is built. Therefore, knowledge of the laws of optimal nutrition can ensure human health. This knowledge is simple and is as follows: consume as much energy as you spend. The energy value (calorie content) of the daily diet should correspond to the daily energy expenditure. Another is the maximum variety of food, which will provide a variety of chemical composition of food to the physiological needs of a person in nutrients (about 600 items). The food consumed should contain proteins, fats, carbohydrates, vitamins, mineral salts, water, fiber, enzymes, flavoring and extractive substances, minor components - bioflavonoids, indoles, anthocyanides, isoflavones and many others. In case of insufficiency of at least one of these components, serious health problems are possible. And to prevent this from happening, a person's daily diet should include approximately 32 different food items.

The optimal ratio of nutrients entering the body contributes to the preservation of health and longevity. But, unfortunately, the majority of the world's population is characterized by a deficiency of the following nutrients: complete (animal) proteins; polyunsaturated fatty acids; vitamins C, B, B2, E, folic acid, retinol, beta-carotene and others; macro- and microelements: Ca, Fe, Zn, F, Se, I and others; dietary fiber. And excessive consumption of such animal fats and easily digestible carbohydrates.

The deficit of protein intake for the majority of the population is on average 20%, the content of most vitamins and microelements is 15-55% less than the calculated values ​​of their need, and dietary fiber is 30% lower. Violation of the nutritional status inevitably leads to poor health and, as a result, to the development of diseases. If we take the entire population of the Russian Federation as 100%, only 20% will be healthy, people in a state of maladaptation (with reduced adaptive resistance) - 40%, and in a state of pre-illness and illness - 20% each, respectively.

Among the most common nutritionally dependent diseases are the following: atherosclerosis; hypertonic disease; hyperlipidemia; obesity; diabetes; osteoporosis; gout; some malignant neoplasms.

The dynamics of demographic indicators in the Russian Federation and Ukraine over the past 10 years is also characterized by exclusively negative trends. The death rate is almost twice the birth rate, life expectancy is significantly inferior not only to developed countries ...

In the structure of causes of death, the leading place is occupied by pathologies of the cardiovascular system and oncological diseases - diseases, the risk of which, among other reasons, depends on malnutrition.

The food shortage in the world should also be taken into account. During the 20th century, the world's population increased from 1.5 to 6 billion people. It is assumed that by 2020 it will grow to 8 billion or more - depending on who counts and how. It is clear that the main issue is the nutrition of such a number of people. Despite the fact that agricultural production has increased by an average of 2.5 times over the past 40 years due to selection and improvement of agronomic methods, its further growth seems unlikely. This means that the rate of agricultural food production in the future will increasingly lag behind the rate of population growth.

A modern person consumes about 800 g of food and 2 liters of water per day. Thus, in just a day, people eat more than 4 million tons of food. Already, the world food shortage exceeds 60 million tons, and the forecasts are disappointing...

Solving the problem of increasing food production by the old methods is no longer possible. In addition, traditional agricultural technologies are not renewable: over the past 20 years, humanity has lost over 15% of the fertile soil layer, and most of the soils suitable for cultivation are already involved in agricultural production.

An analysis of the situation that has developed in recent years in the agro-industrial complex of Russia indicates a decrease in the living population and a drop in the production of all types of agricultural products by more than 1.5 times. With the remaining total volumes of natural and labor resources, the crisis caused a sharp deterioration in the use of arable land, a decrease in the productivity of agroecosystems, more than 30 million hectares of highly productive agrocenoses were taken out of circulation.

Measures taken so far to stabilize the situation on the agricultural market have proved to be ineffective and insufficient. And food imports have exceeded all reasonable limits and called into question food security.

Based on the importance of optimizing the nutrition structure for the health of the nation, development and security of the country, a priority direction has been developed to improve the nutrition of the Russian population: elimination of the deficiency of high-grade protein; elimination of micronutrient deficiencies; creation of conditions for optimal physical and mental development of children; ensuring the safety of domestic and imported food products; increasing the level of knowledge of the population in matters of healthy nutrition. The scientific basis of the modern food production strategy is the search for new resources that provide the optimal ratio of chemical components of food for the human body. The solution to this problem primarily lies in the search for new sources of protein and vitamins.

For example, a plant containing a complete protein, which is not inferior to animal proteins in terms of a set of amino acids, is soybean. The introduction of products from it into the diet makes it possible to compensate for the deficiency of protein, as well as various minor components, in particular, isoflavones.

One of the solutions to the food problem is the chemical synthesis of food products and their components, and some progress has already been made in the production of vitamin preparations. A very promising and already used method of obtaining high-grade food products is their enrichment with protein and vitamins during technological processing, that is, the production of food with a given chemical composition.

Another way is the use of microorganisms as separate components of food products, because the growth rate of microorganisms is a thousand times higher than the growth rate of agricultural animals and 500 times that of plants.

It is important that there is the possibility of directed genetic predetermination of microorganisms in their chemical composition, its improvement, which directly determines their nutritional value and prospects for use.

Thus, in the coming century, food production will not be able to do without the use of high modern technologies and, in particular, without the use of biotechnology, the use of microorganisms to produce food products.

With the growing awareness of the importance of a healthy lifestyle, the demand for food products that do not contain harmful substances has increased. And here DNA technologists could not but participate.

Above, we have already mentioned sugar beets, which produce fructan, a low-calorie substitute for sucrose. This result was obtained by inserting into the beet genome a gene from the Jerusalem artichoke, which encodes an enzyme that converts sucrose into fructan. Thus, 90% of the accumulated sucrose in transgenic beet plants is converted into fructan.

Another example of work on the creation of "functional food" products is an attempt to create decaffeinated coffee. A team of scientists in Hawaii has isolated the gene for the enzyme xanthosine-N7-methyltransferase, which catalyzes the critical first step in caffeine synthesis in coffee leaves and beans. With the help of Agrobacterium, an antisense version of this gene was inserted into Arabica coffee tissue culture cells. Studies of transformed cells have shown that the level of caffeine in them is only 2% of normal. If the work on the regeneration and propagation of transformed plants is successful, then their use will allow avoiding the process of chemical decaffeination of coffee, which will not only save $ 2.00 per kilogram of coffee (the cost of the process), but also preserve the taste of the drink spoiled in this way, which is partially lost during decaffeination. .

Developing countries, where hundreds of millions of people are hungry, are in particular need to improve the quality of food. For example, leguminous plants grown around the world are deficient in certain sulfur-containing amino acids, including methionine. Active attempts are now being made to increase the concentration of methionine in legumes. In GM plants, it is possible to increase the content of storage protein by 25% (this has been done so far for some varieties of beans). Another example already mentioned is the "golden rice" enriched with beta-carotene obtained by Prof. Potrykus from the Technical University in Zurich. Getting an industrial grade would be an outstanding achievement. Attempts are also being made to enrich rice with vitamin B, the lack of which leads to anemia and other diseases.

The work on improving the quality characteristics of crop products well illustrates the possibilities of modern DNA technologies in solving a wide variety of problems.

The term "biotechnology" refers to a set of industrial methods that use living organisms and biological processes for production. Biotechnological techniques are as old as the world - winemaking, baking, brewing, cheese making are based on the use of microorganisms and also belong to biotechnologies.

Modern biotechnology is based on cellular and genetic engineering, which makes it possible to obtain valuable biologically active substances - antibiotics, hormones, enzymes, immunomodulators, synthetic vaccines, amino acids, and food proteins, to create new plant varieties and animal breeds. The main advantage of applying new approaches is to reduce the dependence of production on natural resources, the use of the most environmentally and economically beneficial ways of managing the economy.

The creation of genetically modified plants makes it possible to repeatedly accelerate the process of breeding cultivars, as well as to obtain crops with properties that cannot be bred using traditional methods. Genetic modification of agricultural crops gives them resistance to pesticides, pests, diseases, reducing losses during cultivation, storage and improving product quality.

What is typical for the second generation of transgenic crops that are already being produced on an industrial scale? They have higher agrotechnical characteristics, that is, greater resistance to pests and weeds, and therefore higher yields.

From the point of view of medicine, the important advantages of transgenic products are that, firstly, it was possible to significantly reduce the residual amount of pesticides, which made it possible to reduce the chemical load on the human body in an unfavorable environmental situation. Secondly, to give insecticidal properties to plants, which leads to a decrease in their damage by insects, and this greatly reduces the infestation of grain crops by mold fungi. It is known that they produce mycotoxins (in particular, fumonisins - natural contaminants of cereals), toxic to humans.

Thus, GM products of both the first generation and the second generation have a positive impact on human health not only indirectly - through the improvement of the environment, but also directly - through the reduction of the residual amount of pesticides and the content of mycotoxins. It is not surprising that the area occupied by transgenic crops is increasing year by year.

But now the greatest attention will be paid to the creation of third-generation products with improved or modified nutritional value, resistant to climatic factors, soil salinity, as well as having a prolonged shelf life and improved taste properties, characterized by the absence of allergens.

For crops of the fourth generation, in addition to the above qualities, a change in the architecture of plants (for example, short stature), a change in the time of flowering and fruiting, which will make it possible to grow tropical fruits in the middle zone, a change in the size, shape and number of fruits, an increase in the efficiency of photosynthesis, the production of nutrients with an increased level of assimilation, that is, better absorbed by the body.

Improving methods of genetic modification, as well as deepening knowledge about the functions of food and metabolism in the human body, will make it possible to produce products designed not only to provide good nutrition, but also to further promote health and prevent diseases.

One of the promising areas of plant DNA technologies is the creation of bioreactor plants capable of producing proteins needed in medicine, pharmacology, etc. The advantages of bioreactor plants include the absence of the need for feeding and maintenance, the relative ease of creation and reproduction, and high productivity. In addition, foreign proteins do not cause immune responses in plants, which is difficult to achieve in animals.

There is a need to obtain a whole set of biologically active proteins, which, due to the very low level of synthesis in specific tissues or products, are not available for study on the mechanism of action, wide use or identification of additional applications. Such proteins include, for example, lactoferrin, which is found in a small amount in the milk of mammals, blood leukocytes.

Human lactoferrin (hLF) is promising to be used as a dietary supplement and a therapeutic drug for the prevention and treatment of infectious diseases of the gastrointestinal tract in young children, increasing the body's immune response in malignant and a number of viral (AIDS) diseases. Obtaining lactoferrin from cattle milk, due to its low content, leads to a high cost of the drug. Introduction of lactoferrin gene cDNA into tobacco cells resulted in a number of callus tissues synthesizing truncated lactoferrin, the antibacterial properties of which were much stronger than those of native lactoferrin. The concentration of this truncated lactoferrin in tobacco cells was 0.6-2.5%.

Genes are inserted into the plant genome, the products of which induce an immune response in humans and animals, for example, to the envelope proteins of pathogens of various diseases, in particular, cholera, hepatitis, diarrhea, as well as to antigens of the plasma membranes of some tumors.

Transgenic plants are being created that carry genes that produce certain hormones necessary for human hormone therapy, and so on.

An example of the use of plants to create vaccines is the work done at Stanford University. In the work, antibodies to one of the forms of cancer were obtained using a modernized tobacco mosaic virus, in which the hypervariable region of lymphoma immunoglobulin was inserted. Plants infected with the modified virus produced antibodies of the correct conformation in sufficient quantities for clinical use. 80% of the mice that received the antibodies survived the lymphoma, while all of the mice that did not receive the vaccine died. The proposed method makes it possible to quickly obtain patient-specific antibodies in sufficient quantities for clinical use.

There are great prospects for the use of plants for the production of antibodies. Kevin Uzil and co-workers showed that antibodies produced by soy effectively protected mice from infection with the herpes virus. Compared to antibodies produced in mammalian cell cultures, antibodies produced by plants had similar physical properties, remained stable in human cells, and did not differ in their ability to bind and neutralize the virus. Clinical trials have shown that the use of tobacco-produced antibodies effectively prevented the proliferation of caries-causing mutant streptococci.

A potato-produced vaccine against insulin-dependent diabetes was developed. Potato tubers accumulated a chimeric protein consisting of the B subunit of cholera toxin and proinsulin. The presence of the B subunit facilitates the uptake of this product by cells, which makes the vaccine 100 times more effective. Feeding tubers with microgram amounts of insulin to diabetic mice slowed the progression of the disease.

By his actions, man interfered in the course of the evolutionary development of life on Earth and destroyed the existence of the biosphere, independent of man. But he failed to cancel the fundamental laws governing the biosphere and free himself from their influence.

Reviving after the next cataclysm from the remaining centers, adapting and evolving, life, nevertheless, at all times had the main direction of development. It was determined by the law of Roulier's historical development, according to which, within the framework of the progress of life and the irreversibility of evolution, everything strives for independence from environmental conditions. In the historical process, this desire is realized by complicating the organization, which is expressed in an increase in the differentiation of structure and functions. Thus, at each next turn of the spiral of evolution, organisms appear with an increasingly complex nervous system and its center - the brain. 19th century evolutionary scientists called this direction of evolution "cephalization" (from the Greek "cephalon" - brain) However, the cephalization of primates and the complication of their organism eventually put humanity as a biological species on the verge of extinction according to the biological rule of accelerating evolution, according to which the complication of a biological system means a reduction in the average duration of existence species and an increase in the rate of its evolution. For example, the average lifespan of a bird species is 2 million years, mammals - 800 thousand years, human ancestral forms - 200-500 thousand years. The modern human subspecies exists, according to some ideas, only from 50 to 100 thousand years, but many scientists believe that its genetic capabilities and reserves have been exhausted (Dlekseenko, Keisevich, 1997).

The ancestors of modern man stepped on the path that intensifies the confrontation with the biosphere and leads to disaster about 1.5-3 million years ago, when they first began to use fire. From that moment on, the paths of man and the biosphere diverged, their confrontation began, the result of which may be the collapse of the biosphere or the disappearance of man as a species.

Humanity cannot refuse any of the achievements of civilization, even if they are disastrous: unlike animals that use only renewable energy sources, and in quantities adequate to the ability of the biosphere to self-reproduce biomass, humanity can exist using not so much renewable as non-renewable energy carriers and energy sources. New inventions in this area only increase this opposition.

One of the newest directions in the use of transgenic plants is their use for phytoremediation - purification of soils, groundwater, etc. - from pollutants: heavy metals, radionuclides and other harmful compounds.

Environmental pollution by natural substances (oil, heavy metals, etc.) and synthetic compounds (xenobiotics), often toxic to all living things, is increasing from year to year. How to prevent further pollution of the biosphere and eliminate its existing sources? One of the ways out is the use of genetic technologies. For example, living organisms, primarily microorganisms. This approach is called "bioremediation" - biotechnology aimed at protecting the environment. Unlike industrial biotechnologies, the main goal of which is to obtain useful metabolites of microorganisms, the fight against pollution is inevitably associated with the "release" of microorganisms into the environment, which requires an in-depth understanding of their interaction with it. Microorganisms produce biodegradation - the destruction of hazardous compounds that are not a common substrate for most of them. Biochemical pathways for the degradation of complex organic compounds can be very long (for example, naphthalene and its derivatives are destroyed by a dozen different enzymes).

The degradation of organic compounds in bacteria is most often controlled by plasmids. They are called degradation plasmids, or D-plasmids. They decompose compounds such as salicylate, naphthalene, camphor, octane, toluene, xylene, biphenyl, etc. Most D-plasmids were isolated in soil strains of Pseudomonas bacteria. But other bacteria also have them: Alcalkjenes, Flavobacterium, Artrobacter, etc. Many pseudomonads have plasmids that control resistance to heavy metals. Almost all D-plasmids, as experts say, are conjugative, i.e. capable of self-transportation into the cells of a potential recipient.

D-plasmids can control both the initial stages of the destruction of an organic compound and its complete decomposition. The first type is the OST plasmid, which controls the oxidation of aliphatic hydrocarbons to aldehydes. The genes contained in it control the expression of two enzymes: hydroxylase, which converts hydrocarbons to alcohol, and alcohol dehydrogenase, which oxidizes alcohol to aldehyde. Further oxidation is carried out by enzymes, for the synthesis of which the genes of chromosomes are “responsible”. However, most D-plasmids belong to the second type.

Mercury-resistant bacteria express the mer A gene encoding a mercury transport and detoxification protein. The modified construction of the mer A gene was used to transform tobacco, rapeseed, poplar, and Arabidopsis. In hydroponic culture, plants with this gene were extracted from the aquatic environment up to 80% of mercury ions. At the same time, the growth and metabolism of transgenic plants were not suppressed. Mercury resistance was passed down through seed generations.

During the introduction of three modified mer A gene constructs into the tulip tree (Liriodendron tulipifera), plants of one of the resulting lines were characterized by a rapid growth rate in the presence of dangerous concentrations of mercury chloride (HgCI 2 ) for control plants. Plants of this line absorbed and converted to a less toxic elemental form of mercury and volatilized up to 10 times more ionic mercury than control plants. Scientists believe that elemental mercury vaporized by transgenic trees of this species will immediately dissipate into the air.

Heavy metals are an integral part of land pollutants used in agricultural production. In the case of cadmium, it is known that most plants accumulate it in the roots, while some plants, such as lettuce and tobacco, accumulate it mainly in the leaves. Cadmium enters the soil mainly from industrial emissions and as an impurity in phosphate fertilizers.

One of the approaches to reduce the intake of cadmium in the human and animal organisms can be the production of transgenic plants that accumulate a smaller amount of this metal in the leaves. This approach is valuable for those plant species whose leaves are used for food or animal feed.

You can also use metallothioneins - small cysteine-rich proteins that can bind heavy metals. Mammalian metallothionein has been shown to be functional in plants. Transgenic plants expressing metallothionein genes were obtained, and it was shown that these plants were more resistant to cadmium than control ones.

Transgenic plants with the mammalian hMTII gene had a 60-70% lower concentration of cadmium in the stems compared to the control, and the transfer of cadmium from the roots to the stems was also reduced - only 20% of the absorbed cadmium was transported to the stems.

Plants are known to accumulate heavy metals by extracting them from soil or water. Phytoremediation, subdivided into phytoextraction and rhizofiltration, is based on this property. Phytoextraction refers to the use of fast growing plants to extract heavy metals from the soil. Rhizofiltration is the absorption and concentration of toxic metals from water by plant roots. Plants that have absorbed metals are either composted or burned. Plants differ markedly in their storage capacity. Thus, Brussels sprouts can accumulate up to 3.5% lead (from the dry weight of plants), and its roots - up to 20%. This plant also successfully accumulates copper, nickel, chromium, zinc, etc. Phytoremediation is also promising for the purification of soil and water from radionuclides. But toxic organic compounds are not decomposed by plants; it is more promising to use microorganisms here. Although some authors insist on a decrease in the concentration of organic contaminants during phytoremediation, they are mainly destroyed not by plants, but by microorganisms living in their rhizosphere.

The symbiotic nitrogen-fixing alfalfa Rhlzobium melitotj was introduced with a number of genes that decompose gasoline, toluine and xylene contained in the fuel. The deep root system of alfalfa allows you to clean the soil contaminated with oil products to a depth of 2-2.5 meters.

It should be remembered that most of the xenobiotics appeared in the environment in the last 50 years. But in nature there are already microorganisms capable of utilizing them. This suggests that in populations of microorganisms, genetic events occur fairly quickly, which determine their evolution, more precisely, microevolution. Since there are more and more xenobiotics due to our technogenic civilization, it is important to have a general idea of ​​the metabolism of microorganisms and their metabolic capabilities. All this required the development of a new science - metabolomics. It is based on the fact that bacteria can acquire the ability to process new compounds as a result of mutations. As a rule, this requires several successive mutations or the insertion of new gene systems from those already existing in other types of microorganisms. For example, the decomposition of a stable organohalogen compound requires genetic information found in the cells of various microorganisms. In nature, such information exchange occurs due to horizontal gene transfer, and in laboratories, DNA technology methods taken from nature are used.

Further development of phyto- and bioremediation is a complex problem associated, in particular, with the use of plants and rhizospheric microorganisms. Plants will successfully extract heavy metals from the soil, and rhizospheric bacteria will decompose organic compounds, increasing the efficiency of phytoremediation, promoting plant growth, and plants - the development of microorganisms living on their roots.

Environmental pollution can be considered a disease of ecosystems, while bioremediation can be considered a treatment. It should also be considered as a prevention of numerous human diseases caused by environmental pollution. Compared to other cleaning methods, this one is much cheaper. With diffuse pollution (pesticides, oil and oil products, trinitrotoluene, which pollutes numerous lands), it has no alternative. In cleansing the environment from pollution, it is important to correctly prioritize, minimizing the risks associated with this or that pollution, and taking into account the properties of a particular compound and its impact primarily on human health. Legislative acts and rules are needed to regulate the introduction into the environment of GM microorganisms, with which there are special hopes for purification from any pollutants. Unlike industrial biotechnology, where all parameters of the technological process can be strictly controlled, bioremediation is carried out in an open system, where such control is difficult. To a certain extent, it is always "know-how", a kind of art.

The advantage of microorganisms in the purification of oil products was fully demonstrated when, after the catastrophe of a tanker, 5000 m 3 of oil spilled into the sea off the coast of Alaska. About 1.5 thousand km of the coastline turned out to be contaminated with oil. 11 thousand workers and a variety of equipment were involved in mechanical cleaning (it cost $ 1 million per day). But there was another way: in parallel, to clean the coast, nitrogen fertilizer was introduced into the soil, which accelerated the development of natural microbial communities. This accelerated the decomposition of oil by 3-5 times. As a result, pollution, the consequences of which, according to calculations, could affect even after 10 years, was completely eliminated in 2 years, spending less than $ 1 million on bioremediation.

The development of bioremediation, technologies and methods of its application require an interdisciplinary approach and cooperation of specialists in the field of genetics and molecular biology, ecology, and other disciplines. Thus, the directions of using genetic engineering are very diverse and extensive, and some of them are fantastic and at the same time very promising in terms of achievable results.

The study of the response of living organisms to environmental changes is extremely important for assessing the impact of these changes, especially those of anthropogenic origin, on biodiversity, the conservation of which is the most important task of human civilization.

According to the Organization for Economic Co-operation and Development (OECD), the potential market for bioremediation is more than $75 billion. The accelerated adoption of biotechnologies for environmental protection is due, in part, to the fact that they are much cheaper than other cleaning technologies. According to the OECD, bioremediation is of local, regional and global importance, and both natural organisms and GMOs will increasingly be used for purification.

Given the limited reserves of fossil energy, special attention should now be paid to the possibility of using new types of fuel - methane, hydrogen, etc., as well as renewable energy sources. However, in the overall energy balance, such environmentally friendly energy sources as the energy of the Sun, sea currents, water, wind, etc., can make up no more than 20% of their total production. In this situation, one of the most promising renewable energy sources is biomass, the methods of using which are constantly being improved. At the same time, along with direct combustion, bioconversion processes are widely used, for example, alcoholic and anaerobic fermentation, thermal conversion, gasification, pyrolysis, etc. used as a fuel additive to replace imported oil. For the same purpose, the exploitation of natural thickets of black willow grass, which occupies about 6 million hectares in the northeastern regions of the country, has begun.

If in India, China and some other countries agricultural waste is disposed of in order to obtain biogas, then in Sweden, Germany, Brazil, the USA, Canada, agricultural crops are specially grown for the production of ethanol fuel alcohol. An effective substitute for fossil fuels is rapeseed and colza oil, the spring forms of which can be cultivated in Russia up to the Arctic Circle. Soybean, sunflower and other crops can also be a source of vegetable oils for biofuel production. Brazil is increasingly using sugar cane to produce fuel ethanol, and corn is being used more and more in the United States.

The energy return coefficient (the ratio of the total energy equivalent of useful products to all energy costs for its production) is for sugar beet - 1.3; fodder grasses - 2.1; rapeseed - 2.6; wheat straw - 2.9. At the same time, due to the use of 60 centners of wheat straw from each hectare as a feedstock, it is possible to obtain 10 thousand m 3 of generator gas, or 57.1 GJ.

Due to the rapid depletion of natural resources of oil, gas and coal in many countries, special attention is paid to the so-called oil-bearing plants - Euphorbia lathyris (oil spurge) and E.tirucallii from the spurge family (Kupharbiacea), containing latex, the composition of terpenes of which approaches in its characteristics to high quality oil. At the same time, the dry mass yield of these plants is about 20 t/ha, and the yield of an oil-like product in the conditions of Northern California (i.e., in the zone of 200-400 mm of precipitation per year) can reach 65 barrels of raw materials per 1 ha. Therefore, it is more profitable to grow vegetable substitutes for fossil fuels, since more than 3,600 petrodollars can be obtained from each hectare, which in grain equivalent will be 460 c/ha, i.e. 20 times the average wheat yield in the US and Canada. If we recall the well-known US slogan “for every barrel of oil, a bushel of grain”, then at today's prices for oil, gas and grain, this means an exchange of 1 grain dollar for approximately 25 petrodollars. Of course, a barrel of oil will not replace a bushel of grain in the literal sense, and not every zone will be able to cultivate these types of plants. But obtaining alternative fuels through targeted plant breeding also turns the technogenic-energy component of highly productive agrophytocenoses into a reproducible and environmentally friendly factor in the intensification of crop production, and, of course, this is one of the most painless ways out for such states as Ukraine - to use plants on an increasing scale in as renewable resources, including energy (biodiesel fuel, lubricants, etc.). For example, the production of winter rapeseed already provides a ratio of energy consumption and energy output of 1:5.

The fundamental point of the current stage of breeding is a clear understanding that the basis for its development, including the use of genetic engineering techniques, is biodiversity.

The evolution of the plant kingdom followed the path of multiplication of the number of species and their "ecological specialization". This fact indicates the danger of a decrease in biological (genetic) diversity in the biosphere in general and in agroecosystems in particular. The sharp narrowing of species and genetic diversity has reduced not only the resistance of crop production to the vagaries of weather and climate change, but also the ability to more efficiently utilize solar energy and other inexhaustible natural resources (carbon, oxygen, hydrogen, nitrogen and other biophilic elements), which, as are known to make up 90-95% of the dry matter of the phytomass. In addition, this leads to the disappearance of genes and gene combinations that could be used in the breeding work of the future.

One and the same area, emphasized Ch. Darwin (1859), can provide the more life, the more diverse the forms inhabiting it. Each cultivated plant species, due to its evolutionary history and the specific work of the breeder, is characterized by its own “agroecological passport”, i.e. the confinement of the size and quality of the crop to a certain combination of temperature, humidity, lighting, the content of mineral nutrition elements, as well as their uneven distribution in time and space. Therefore, the decrease in biological diversity in agrolandscapes also reduces the possibility of differentiated use of natural environment resources, and, consequently, the implementation of differential land rent of types I and II. At the same time, the ecological stability of agroecosystems is also weakened, especially in unfavorable soil, climatic and weather conditions.

The scale of the disaster caused by the defeat of potatoes by phytophthora and nematodes, the catastrophic losses of wheat due to rust damage, corn due to the epiphytoty of helminthosporiasis, the destruction of cane plantations due to viruses, etc. are known.

A sharp decrease in the genetic diversity of plant species cultivated at the beginning of the 21st century is clearly evidenced by the fact that out of 250 thousand species of flowering plants over the past 10 thousand years, man has introduced into culture 5-7 thousand species, of which only 20 cultures (14 of which belong to cereals and legumes) form the basis of the modern diet of the world's population. In general, to date, about 60% of food is produced due to the cultivation of several grain crops, and over 90% of human needs for food are provided by 15 species of agricultural plants and 8 domesticated animal species. Thus, out of 1940 million tons of grain production, almost 98% is accounted for by wheat (589 million tons), rice (563 million tons), corn (604 million tons) and barley (138 million tons). Of the 22 known species of rice (genus Oryza), only two are widely cultivated (Oryza glaberrima and O. sativa). A similar situation exists with legumes, the gross production of the 25 most important species of which is only about 200 million tons. Most of them are soybeans and peanuts, cultivated mainly as oilseeds. For this reason, the diversity of organic compounds in the human diet has significantly decreased. It can be assumed that for Homo sapiens, as one of the biological species, the need for a high biochemical variability of food is recorded in the evolutionary "memory". Therefore, the tendency to increase its monotony can have the most negative consequences for health. Due to the wide spread of oncological diseases, atherosclerosis, depression and other diseases, attention is drawn to the lack of vitamins, tonic substances, polyunsaturated fats and other biologically valuable substances.

Obviously, an important factor in the spread of a valuable culture is the scale of its use. Thus, the rapid increase in the area of ​​soybeans and corn in the United States and other countries is due to the production of hundreds of items of the corresponding products. The task of diversification is also very relevant for other crops (for example, high-quality beer has been produced from sorghum, whiskey from rye, etc.).

Greater attention in terms of solving the interrelated problems of healthy food and increasing the species diversity of agroecosystems deserves an increase in the area under crops of such valuable crops as buckwheat (Fagopyrum), which has high adaptive capabilities in various, including adverse environmental conditions, amaranth (Amaranthus), quinoa (Chenopodium quinoa), rapeseed, mustard and even potatoes.

With the development of geographical discoveries and world trade, the introduction of new plant species has also become widespread. Written monuments testify, for example, that as early as 1500 BC. The Egyptian pharaoh Hatshepsut sent ships to East Africa to collect plants used in religious ceremonies. In Japan, there is a monument to Taji Mamori, who, by order of the emperor, traveled to China to collect citrus plants. Agriculture has played a special role in the mobilization of plant genetic resources. It is known from the history of the United States that already in 1897 Niels Hansen arrived in Siberia in search of alfalfa and other fodder plants that could successfully grow in the arid and cold conditions of the prairies of North America. It is believed that it was from Russia at that time that such important fodder crops as brome, pig, fescue, cocksfoot, white bent grass, alfalfa, clover, and many others were introduced into the United States. Around the same time, Mark Carleton was harvesting wheat varieties in Russia, of which the Kharkov variety type occupied more than 21 million acres annually in the United States for a long period and became the basis for durum wheat production in the Northern Plains zone (Zhuchenko, 2004).

The introduction of new plant species into the culture continues at the present time. In the Peruvian Andes, a variety of lupine (tarwi) was discovered, which was eaten by the ancestors of modern Indians, which surpasses even soy in protein content. In addition, tarvi is resistant to low temperatures, undemanding to soil fertility. Breeders managed to obtain forms of tarwi containing less than 0.025% alkaloids versus 3.3% in the original material. Other species of economic value include Australian Grass (Echinochloa lurnerana), which can be an excellent grain crop to match millet in very dry areas. Among promising crops, the Bauhinia esculenta species deserves attention, which, like Psophocarpus tetragonolobus, forms tubers, and its seeds contain more than 30% protein and fat. In very dry conditions, Voandzeia subterranea can be used, which is not only rich in protein, but also more drought tolerant than peanuts, and better resistant to diseases and pests. For dry and infertile oilseed lands, Cucurbita foetidissima from the Cucurbitaceae family is considered promising, and for saline pasture lands, some species of Atriplex genus from the Chenopodiaceae family, which excrete excess salt through the leaves, are considered promising.

Currently, in many countries of the world, active breeding work is underway with amaranthus (Amaranthus), a forgotten culture of the Incas, in the seeds of which, compared to the used cereal species of plants, contains twice as much protein, including 2-3 times more lysine and methionine, 2-4 times more fat and so on. Corn lines have been found that, due to the presence of Spirillum lipoferum bacteria on their roots, fix atmospheric nitrogen in the same amount as soybean plants. It was found that nitrogen-fixing bacteria also function on the roots of a number of species of tropical grasses, assimilating nitrogen no less actively than bacteria of the genus Rhizobium in legumes. So, it was possible to find species of tropical grasses that can fix up to 1.7 kg of nitrogen per day per 1 ha, i.e. 620 kg/year.

In many countries, including European countries, potatoes are the main source of vitamin C, as they are consumed in large quantities. It is known that the production of potatoes in the world is about 300 million tons.

At the same time, out of 154 known potato species, only one, Solanum tuberosum, has become widespread. Obviously, due to the increased possibilities of breeding to increase the potential productivity of plants, as well as the need to increase the environmental sustainability of agrocenoses and the development of territories unsuitable for crop production, the scale of human activity to introduce new plant species into cultivation will increase significantly. Ultimately, "unconscious" (Darwin's term) and conscious selection led to the fact that the adaptive potential of cultivated plants differs significantly from that of their wild ancestors, not only due to differences in the adaptability criteria themselves, but also in terms of its main components: potential productivity, resistance to abiotic and biotic stresses, the content of economically valuable substances.

Along with the preservation of the plant gene pool in nature reserves, wildlife sanctuaries and national eco-parks, i.e. in situ settings, the establishment of “gene banks” or “germplasm banks” to ensure the safe conservation of ex situ collections will play an increasingly important role in the coming period. The initiator of the organization of the latter was N.I. Vavilov, who collected in VIR the largest bank of plant resources in the world at that time, which served as an example and basis for all subsequent banks, and most importantly, saved a number of countries from devastation and hunger more than once (for example, due to the presence of resistance genes in the VIR genebank).

Thanks to the continuation of the ideology of N.I. Vavilov, by the end of the 1990s, national and international plant collections included more than 6 million samples, including more than 1.2 million cereals, 400 thousand food legumes, 215 thousand fodder, 140 thousand vegetables, over 70 thousand . root crops. At the same time, 32% of samples are stored in Europe, 25% - in Asia, 12% - in North America, 10% - in Latin America and International Centers, 6% - in Africa, 5% - in the Middle East.

The USA (550 thousand), China (440 thousand), India (345 thousand) and Russia (320 thousand) hold the largest samples of genetic collections in terms of quantity and quality. Along with the conservation of plant resources in genebanks, the creation of natural reserves of flora and fauna is becoming more widespread. Thanks to the sharply increased integration of the world food market, the exchange of plant genetic resources between countries has also increased significantly. These processes are based on the understanding that no country or region is self-sufficient in terms of providing genetic resources. The creation of national botanical gardens in a number of countries contributed greatly to the mobilization of genetic resources. Among them, for example, a botanical garden, created in London in 1760 and constantly importing exotic plant species from colonial countries.

Currently, the International Council for Plant Genetic Resources (IBPGR) is coordinating the work on the conservation of the plant gene pool in the world. Since 1980 the European program of cooperation in the field of genetic resources has been implemented. An important role in this is also played by the FAO Commission on Plant Genetic Resources, the decisions of international conferences, and the Convention on Biological Diversity adopted in 1992. At the same time, gene banks of different types function. Some of them support only one crop and its wild relatives, others - several crops of a certain soil-climatic zone; if some contain basic collections of long-term storage, others are focused on meeting the needs of breeding centers and research institutions. Thus, the gene bank in Kew Gardens (England) stores exclusively wild plants (about 5,000 species).

The adaptive strategy of agricultural intensification puts forward qualitatively new requirements for the mobilization of world plant resources in terms of the collection, storage and use of the gene pool, including the introduction of new plant species into cultivation. Currently, over 25 thousand species of higher plants are under the threat of complete destruction in the world, including in Europe - every third of 11.5 thousand species. Many primitive forms of wheat, barley, rye, lentils and other crops have already been lost forever. Local varieties and weed species are disappearing especially fast. So, if in China and India in the early 50s. 20th century thousands of varieties of wheat were used, then already in the 70s - only dozens. At the same time, each species, ecotype, local variety is a unique complex of co-adapted blocks of genes created during a long natural or artificial selection, which ultimately ensures the most efficient utilization of natural and anthropogenic resources in a particular ecological niche.

Understanding the retrospective nature of the evolutionary "memory" of higher plants clearly indicates the need to preserve the species diversity of the flora not only in gene banks and centers of genetic resources, but also in natural conditions, i.e. in a state of constantly evolving dynamic system. At the same time, the creation of genetic collections of genetic systems for the transformation of genetic information, including res-systems, mei-mutants, gametocidal genes, polyploid structures, various types of recombination systems, reproductive isolation systems, etc., deserves much more attention. It is clear that they can be essential for the development selection of the future using genetic engineering technologies. It is also important to identify and preserve the genetic determinants of the formation of stable homeostatic systems, synergistic, cumulative, compensatory and other coenotic reactions that provide ecological "buffer" and dynamic balance of the biocenotic environment. More attention should be paid to such genetically determined traits of plants as competitiveness, allelopathic and symbiotic interactions, and other environmental effects realized at the biocenotic level. Particular attention should be paid to plant species with constitutive resistance to environmental stressors. It is known that in the second half of the XX century. in a number of countries, the area under this type of crops has increased significantly (sometimes 60-80 times).

Currently, there are more than 1,460 national gene banks in the world, including about 300 large ones, which provide guaranteed storage of samples of cultivated plants and their wild relatives under ex situ conditions. Ex situ collections are also kept by botanical gardens, of which there are about 2,000 in the world (about 80,000 plant species, 4 million specimens, and 600 seed banks). Their presence is a sign of national sovereignty, the level of culture, concern for the future of the country and the world. By 2002, over 532,000 plant specimens were preserved in international centers under the control of the FDO advisory group, of which 73% belong to traditional and landrace varieties, as well as wild relatives of cultivated plants. As Dleksanyan (2003) points out, a distinction should be made between the concepts of “genebank” and “ex silu collections”. If the first is the guaranteed storage of the gene pool in specially equipped facilities, then "ex situ collections" include accessions that are of interest to their holders.

In the early 50s. In the 20th century, the first semi-dwarf rice variety was obtained by using the dwarfing gene of the Chinese variety Fee-geo-woo, and the Gaines wheat variety in the irrigated lands of the Pacific Northwest of the United States produced a record yield of 141 c/ha. In 1966, the variety IR 8 was created, which received the nickname "miracle rice". With high agricultural technology, these varieties produced 80 and even 130 q/ha. Similar results were obtained for millet. If the yield index for old varieties was 30-40%, then for new varieties it was 50-60% and higher.

Further opportunities to increase yields by increasing the yield index are limited. Therefore, much more attention should be paid to increasing the value of net photosynthesis. It is necessary to focus on the wide species and varietal heterogeneity of agroecosystems and agrolandscapes in field crop production, along with the selection of insurance crops, as well as mutually insured crops and varieties, and includes a differentiated approach to realizing the adaptive potential of each of them. The high potential productivity of a variety and agroecosystem, achieved by (and sometimes at the expense of) reducing their ecological resistance to environmental factors limiting the size and quality of the crop, as well as the functioning of excessively bioenergy-consuming ecological stability, cannot be considered as adaptive, since for cultivated plants, the main indicator of adaptability in the long run is to ensure the high size and quality of the crop. Gene pools accumulated in genebanks can be a source for scientifically based breeding to create the necessary varieties.

It should be emphasized that millions of samples have been collected in the world's genebanks of cultivated plants, but so far only 1% of them have been studied in relation to their potential properties (Zhuchenko, 2004). At the same time, the control and improvement of their genetic component - the gene pools of agricultural species, which determines the characteristics of local agrosystems, is of paramount importance for the creation of sustainable agrosystems.

Disease-modifying disease-modifying antirheumatic drugs (DMARDs) are a group of drugs commonly used in patients with rheumatoid arthritis (RA). Some of these drugs are also used to treat other conditions, such as and systemic lupus erythematosus. They help reduce pain and inflammation, reduce or prevent joint damage, and maintain joint structure and function.

What are basic antirheumatic drugs

They work to suppress overactive immune or inflammatory systems in the body. They take weeks or months to take effect and are not intended to relieve symptoms immediately.

Other medicines, such as pain relievers (such as ibuprofen or naproxen) and sometimes prednisone, provide faster relief from current symptoms. DMARDs are often used in combination with these drugs to reduce the total amount of medication needed and prevent joint damage.

Disease-modifying antirheumatic drugs

The choice of DMARD depends on a number of factors, including the stage and severity of the general condition, the balance between possible side effects and expected benefits, and patient preference. Before starting treatment, the patient and physician should discuss the benefits and risks of each type of therapy, including possible side effects and toxicity, dosing schedule, frequency of monitoring, and expected results. Certain tests, including blood tests for past exposure to certain infections, may be needed before you start taking some of these medicines.

In some cases, one basic antirheumatic drugs are used. In other cases, more than one medication may be recommended. Sometimes a patient must try different medications or combinations to find the one that works best and has the fewest side effects. A patient who does not fully respond to one DMARD may be given a combination of DMARDs, such as methotrexate plus another drug.

The most common medications are methotrexate, sulfasalazine, hydroxychloroquine, and leflunomide. Less commonly used medications include gold salts, azathioprine, and cyclosporine.

Methotrexate - originally used as a chemotherapy treatment for cancer. When used at much lower doses for rheumatoid arthritis and other rheumatic conditions, methotrexate works to reduce inflammation and reduce joint damage. It is usually taken once a week as a tablet, liquid, or injection. Methotrexate can be combined with other drugs or with the help of biological agents, unless it does not sufficiently control the patient's disease.

Common side effects include stomach upset and mouth pain. Methotrexate can affect the production of blood cells in the bone marrow. A low white blood cell count can cause fever, infections, swollen lymph nodes, and easy bruising and bleeding. Liver or lung damage can occur even at low doses and therefore needs to be monitored. People using methotrexate strongly discourage the consumption of alcoholic beverages due to the increased risk of liver damage from this combination. Patients should not become pregnant while taking methotrexate.

Surveillance reduces the risk of long-term methotrexate damage. Testing is done before starting treatment to determine if certain infections have been found. A chest x-ray is also recommended before starting treatment, and regular blood tests are recommended. While taking methotrexate, all patients should take folic acid 1 mg per day or 5 mg per week to reduce the risk of certain side effects such as upset stomach, sore throat, white blood cell count, and abnormal liver function.

Sulfasalazine is used in the treatment of rheumatoid arthritis and arthritis associated with ankylosing spondylitis and inflammatory bowel disease (ulcerative colitis and Crohn's disease). It is not clear how sulfasalazine works. It may be combined with other basic antirheumatic drugs if a person does not respond adequately to one drug. It is taken as a tablet two to four times a day and is usually started at a low dose and increased slowly to minimize side effects.

Side effects of sulfasalazine include changes in blood composition, nausea or vomiting, sensitivity to sunlight, skin rashes, and headaches. People who are allergic to sulfa drugs such as sulfamethoxazole-trimethoprim may cross-react with sulfasalazine and should therefore not take it. Periodic monitoring of blood counts at regular intervals is recommended.

Sulfasalazine - yellow-orange color; patients who take it may notice that their urine, tears, and sweat develop an orange tint that can stain clothing and contact lenses. Patients should drink plenty of fluids while taking sulfasalazine and avoid taking it on an empty stomach or with antacids.

Hydroxychloroquine Originally developed as a treatment for malaria, it was later discovered to improve the symptoms of arthritis. It can be used early in RA and is often used in combination with DMARDs. It is also very commonly used for healing. It can be combined with steroid medications to reduce the amount of steroid needed. It is usually taken as a tablet once or twice a day.

Taking a high dose of hydroxychloroquine for long periods of time may increase the risk of damage to the retina, although high doses are not usually required to treat rheumatoid conditions or lupus. It is recommended to have an eye examination by an ophthalmologist before starting treatment and periodically thereafter. An eye examination is usually performed once a year.

Leflunomide - inhibits the production of inflammatory cells to reduce inflammation. It is often used alone, but may be used in combination with methotrexate for people who have not responded adequately to methotrexate alone or together with a biological agent. Taken orally once a day.

Side effects include rashes, temporary hair loss, liver damage, nausea, diarrhea, weight loss, and abdominal pain. Testing for prior exposure to hepatitis and regular blood counts during therapy are necessary to monitor for liver damage and other toxic events. Patients should not become pregnant while taking leflunomide or while it is still present in the body.

Azathioprine has been used in the treatment of cancer, RA, lupus and several other inflammatory diseases since the 1950s. It has also been used in organ transplants to prevent rejection of the transplanted organ. Azathioprine is usually reserved for patients who have not responded to other treatments.

The most common side effects are nausea, vomiting, decreased appetite, liver function abnormalities, low white blood cell counts, and infection. Usually taken orally once every four hours daily. A blood test is recommended during treatment with azathioprine.

Cyclosporine was originally developed to prevent rejection after organ transplants. It works in patients with rheumatoid arthritis to suppress T-lymphocytes, a cell that promotes inflammation associated with rheumatoid arthritis. There is concern about the long-term safety of ciclosporin and its association with kidney disease and high blood pressure, so it is usually reserved for patients who have not responded to other treatments. It is usually taken orally as a tablet or liquid form twice a day; an injectable form is also available. It is sometimes used to treat kidney disease due to lupus.

Side effects include high blood pressure, swelling, kidney damage, increased hair growth, nausea, diarrhea, and heartburn. Patients should regularly monitor blood pressure and kidney function.

Biological agents

Another class of drugs used in people with and associated inflammatory diseases are biological agents. They are sometimes referred to as biologic DMARDs, including etanercept, adalimumab, infliximab, which are part of a class of drugs called tumor necrosis factor (TNF) inhibitors, and a number of other agents with different targets, including anakinra, abatacept, rituximab, and tocilizumab. Another group of DMARDs called kinase inhibitors includes tofacitinib. A biological DMARD or kinase inhibitor is often combined with methotrexate or other medications to increase effectiveness.

Treatment of rheumatoid arthritis with disease-modifying disease-modifying antirheumatic drugs (DMARDs)

In the treatment of rheumatoid arthritis, drugs are used that slow the progression of joint erosion. These are disease-modifying disease-modifying antirheumatic drugs (DMARDs) that are an important part of the overall treatment program. What are these drugs and how do they work?

Disease-modifying drugs act on the immune system to slow the progression of rheumatoid arthritis, from which they get their name. The category of DMARDs includes many different drugs, but some of them are used most often:

Rheumatex (Methotrexate)- the main drug of the DMARD category. It works in the same way as other drugs, and in many cases is more effective. It is also relatively inexpensive and mostly safe. Like other DMARDs, methotrexate has a number of side effects: it can cause stomach upset, it can be toxic to the liver or bone marrow, and it can affect pregnancy. In rare cases, it causes difficulty in breathing. When taking methotrexate, good blood circulation is essential. Concurrent use of folic acid may reduce some of the side effects. The most important advantage of methotrexate is the possibility of its use for a long period. The drug can also be given to children.

Biological agents: Enbrel (etanercet), Humira (adalimumab), Kineret (anakinra), Orencia (abatacet), Remicad (infliximab), and Rituxan (rituximab). These are the newest drugs for the treatment of rheumatoid arthritis, administered subcutaneously or intravenously. They neutralize the activity of the immune system that destroys the joints. In combination with methotrexate, these drugs help most people overcome the symptoms of rheumatoid arthritis. According to studies, these drugs have fewer side effects than other DMARDs. One of the complications is an increased susceptibility to acute infectious diseases. These drugs can adversely affect the liver, blood condition and should be used with caution in the presence of chronic heart conditions. Other possible side effects may appear only after long-term use of drugs.

Plaquenil (hydroxychloroquine) And Azulfidine(sulfazalin ) used in moderate rheumatoid arthritis. They are not as effective as other DMARDs, but have fewer side effects. In rare cases, Plaquenil adversely affects the eyes. Patients taking this drug should be examined annually by an ophthalmologist.

Minocin (minocycline)- an antibiotic that can stop the inflammatory process in RA. Its effect is manifested after a few months. In other cases, it takes a year for the full range of side effects to appear. With prolonged use, minocycline can cause skin pigmentation.

Arava (leflunomide) acts like methotrexate and is more effective in combination with it. The drugs have similar side effects. Arava may cause diarrhea, in which case its use should be discontinued. Since Arava has a negative effect on the fetus, it is contraindicated in women during pregnancy.

Neoral (azathioprine) used for various diseases accompanied by inflammation, including rheumatoid arthritis. However, due to its negative effect on kidney function and other side effects, it is usually used to treat flare-ups of rheumatoid arthritis when other drugs have failed.

Imunar (azathioprine) used in various inflammatory conditions, including rheumatoid arthritis. The most typical side effects are nausea and vomiting, sometimes there are stomach pains and diarrhea. Long-term use of azathioprine increases the chance of developing cancer.

DMARDs slow the progression of rheumatoid arthritis and help many people improve their quality of life. In some cases, remission may occur. Basically, drugs provide a slowdown in the progression of the disease.

The use of a single DMARD or a combination of them can prolong the asymptomatic course of rheumatoid arthritis and mitigate the acute manifestations of the disease. Your joints will need less time for the morning "rocking". At your next physical examination, your rheumatologist may tell you that there are no new lesions on your most recent x-rays. Also, regular use of BPRP reduces the likelihood of developing a long-term destructive process in the joints.

Are DMARDs safe? All DMARDs are approved by the US Food and Drug Administration. Many people take these drugs without experiencing any side effects at all.

However, while acting on the symptoms of rheumatoid arthritis, DMARDs affect the entire body, and their powerful effects tend to cause some side effects. There are the following typical side effects of DMARDs:

Stomach upset. DMARDs often cause nausea, sometimes vomiting, and diarrhea. These symptoms can be controlled with other medicines. Complications also disappear as your body gets used to the drug. If the symptoms are too uncomfortable, your rheumatologist will prescribe you another remedy.

Liver dysfunction. This complication is less common than indigestion. You will need regular blood tests to check for liver damage.

Blood condition. DMARDs can interfere with the immune system and increase the risk of infections. It can also reduce the level of white blood cells that protect the body from infections. Low red blood cells (anemia) increase fatigue. A simple test performed regularly will help keep your red blood cell levels in check.

For citation: Badokin V.V. The main symptom-modifying delayed-acting drugs in the treatment of osteoarthritis // RMJ. 2011. No. 12. S. 725

Osteoarthritis (OA) is the main nosological form of degenerative joint diseases. It occurs in more than 70% of patients aged 65 years, and radiological symptoms of this disease are even more often detected. OA involves, first of all, load-bearing (knee and hip) joints into the orbit of its pathological process, and this significantly worsens the quality of life of patients, leads to disability, especially in the elderly. It represents a serious socio-economic problem, being one of the main causes of persistent disability. According to EULAR (2003), the risk of disability due to osteoarthritis of the knee is equal to the risk associated with heart disease and is the 4th leading cause of disability in women and 8th in men. The long-term prognosis of OA in specific patients is difficult to predict, including the course of individual clinical symptoms, the progression of radiographic (structural) changes, and impaired quality of life.

OA is considered as a multifactorial disease, in the development of which various factors (mechanical, hormonal, genetic) take part. The contribution of these factors to the development, individual manifestations and outcome of this disease in individual patients is extremely variable. It is well known that various risk factors are involved in gonarthrosis, coxarthrosis and arthrosis of the small joints of the hands. This made it possible for some authors to consider OA as a heterogeneous group of joint diseases of various etiologies, but with similar biological, morphological and clinical signs and overall outcome. Osteoarthritis is based on an imbalance between anabolic and catabolic processes in the tissues of the joint, and above all in hyaline cartilage - the main and primary springboard for pathological changes. The disease is characterized by a chronic slowly progressive course and leads to a decrease in the volume of hyaline cartilage, up to its complete loss.
The pathogenesis of primary osteoarthritis has been largely deciphered (in particular, the molecular mechanisms of its development). Of decisive importance is attached to chronic overload of the joints, including their micro- and macrotraumatization. This leads to a disruption in the activity of chondroblasts and chondrocytes, and then to insufficient synthesis of proteoglycans by chondrocytes, as well as a quantitative and qualitative disruption in the formation of glycosaminoglycans and proteoglycan aggregates. On the other hand, there are changes in the subchondral bone, its sclerosis develops, which further increases the load on the affected joint. Important is the activation of matrix proteinases (collagenase, phospholipase A2), overexpression of pro-inflammatory cytokines (interleukin-1 and tumor necrosis factor-α), deficiency of anti-inflammatory cytokines, for example, transforming growth factor-ta-β and plasminogen-1 inhibitor, which inhibit anabolic processes in the affected cartilage. A certain role in the pathogenetic cascade of osteoarthritis belongs to superoxide radicals, a decrease in the synthesis of hyaluronic acid by synoviocytes, as well as hyperproduction of prostaglandin E2, which, along with other factors, contributes to inflammation in the tissues of the joint, stimulates the activity of osteoblasts and induces fibroplastic cartilage degeneration.
Pathological changes in osteoarthritis (OA) reflect both damage to the tissues of the joint and the response to this damage. Although the most pronounced changes occur in the articular cartilage, all joint tissues and periarticular soft tissues are involved in the pathological process. In addition to degeneration and reduction in the volume of hyaline cartilage, inflammation of the synovium is observed, as well as bone remodeling with subchondral sclerosis, the formation of osteophytes and subchondral cysts, fibrosis of the articular capsule, meniscus degeneration, and periarticular muscle atrophy. In addition, ligaments, entheses, sensory nerves are involved in the pathological process.
The interest of all the structures that make up the joint, which can be considered as an independent organ, leads to various mechanisms for the onset of pain - one of the leading symptoms of this disease. Thus, damage to the subchondral bone contributes to the development of pain through the occurrence of intraosseous hypertension and microfractures, the formed osteophytes lead to traumatization of sensory nerves, and damage to the periarticular muscles is accompanied by their spasm. However, inflammation is of paramount importance in the origin of pain, which is of paramount importance in the development and progression of OA.
The inflammatory process is localized not only in the synovial membrane, but also in cartilage, bone and periarticular soft tissues, including the articular capsule, ligaments and tendons, which is accompanied by the development of synovitis, chondritis, osteitis and periarthritis, respectively. The multifaceted nature of the lesion in OA has become more evident with the introduction of new technologies into clinical practice, in particular, magnetic resonance imaging (MRI). MRI helps to determine the phenotype of OA, clarify the relationship between pain and structural changes in this disease, visualize the topic of the lesion, and identify targets for therapy. This method allows you to identify morphological changes in various tissues of the joint in the presence of minimal radiographic changes or even in their absence. While little is known about the clinical significance of MRI symptoms, it is nevertheless clear that bone marrow changes are associated with a high rate of radiological progression of OA, and pain correlates with synovitis and bone marrow edema (probably intraosseous hypertension).
The treatment of this disease is complex and includes non-pharmacological, pharmacological and clinical methods. Methods of pharmacotherapy include non-opioid and opioid analgesics (paracetamol, tramadol), systemic non-steroidal anti-inflammatory drugs (NSAIDs), local therapy (capsaicin, NSAIDs, dimexide), the so-called chondroprotectors (simptom-modifying drugs of delayed action), intra-articular injections (glucocorticoids, drugs hyaluronic acid), experimental therapy (biological response modulators, drugs that affect bone metabolism).
Of the symptom-modifying drugs of delayed action, the natural components of the cartilaginous intercellular substance - glucosamine and chondroitin sulfate, which are the most studied among the drugs of this group and more evidence-based, are of paramount importance. They are classified as specific antiarthrotic drugs, which are characterized by a slower development of a symptom-modifying effect, a pronounced aftereffect, when after stopping treatment the effect persists for 4-8 weeks or more, and most importantly, they have potential structural-modifying (chondroprotective) ) properties. Therefore, glucosamine and chondroitin sulfate not only actively affect the main clinical manifestations of this disease (namely, suppress pain and normalize the function of the affected joints), but also slow down the progression of OA, normalize or stabilize structural changes in the hyaline cartilage, and prevent changes in the unaffected joint. (Table 1).
Glucosamine has the most solid evidence base. It is a monosaccharide and a natural component of glycosaminoglycans in the articular matrix and synovial fluid. Glucosamine has a specific effect on osteoarthritic cartilage and stimulates the synthesis of a complete extracellular matrix by chondrocytes, and, above all, its most important component, proteoglycans and hyaluronic acid (Table 2). It significantly reduces the activity of catabolic enzymes in cartilage, including matrix metalloproteinases.
Glucosamine is synthesized from marine-derived chitin and contains several salts. In medical practice, two of its salts are used - sulfate and hydrochloride. Glucosamine sulfate is a pure substance with a molecular weight of 456.46 and is a sulfated derivative of the natural amino monosaccharide glucosamine. It is a normal component of glycosaminoglycans and proteoglycans, as well as a substrate for the synthesis of glycosaminoglycan chains, aggrecan, and other components of cartilage. When taken orally or parenterally, it accumulates in articular cartilage. It is characterized by rapid absorption from the gastrointestinal tract. Absolute bioavailability after the first passage through the liver is 26%. When administered intramuscularly, the concentration of glucosamine sulfate is usually 5 times higher than when taken per os.
In a systematic Cochrane review, which analyzed the most significant studies on the efficacy and tolerability of glucosamine, its symptomatic effect was highly evaluated. The effectiveness of glucosamine was significantly higher than placebo in terms of reducing the intensity of joint pain, improving the Lequesne index, as well as the percentage of patients who responded to therapy. At the same time, no significant results were obtained when comparing the effectiveness of glucosamine and placebo in terms of such parameters as reducing pain on the WOMAC index scale, stiffness, and improving the function of the affected knee joints.
Speaking of glucosamine, it is impossible to avoid two solid studies in which the structure-modifying effect of this drug was registered. In the first of these studies, 212 patients were randomized into 2 groups who received glucosamine sulfate or placebo regularly for 3 years. The width of the joint space increased by the end of the study by 0.12 mm in the main group taking glucosamine sulfate, and in the placebo group it decreased by 0.24 mm. These data indicate not only the symptom-modifying, but also the structural-modifying efficacy of this drug, i.e. its ability to actively influence the rate of progression of OA. However, not all patients with long-term treatment with glucosamine managed to achieve a decrease in the rate of radiographic progression. So, after three years of continuous use of this drug, rapid progression of the disease was observed in 15% of patients, while the narrowing of the joint space exceeded 0.5 mm. Risk factors for such an aggressive course of OA have not yet been identified. It should also be noted that the therapeutic activity of glucosamine is shown only in patients with gonarthrosis, but not coxarthrosis.
Later, the structure-modifying effect of glucosamine was reported by Pavelka et al. . Indirectly, these data are confirmed by the results of a long-term (8-year average) follow-up of patients who were treated with glucosamine in the first 3 years of follow-up. In the next 5 years, 10.2% of patients in the main group and 14.5% in the control group underwent knee arthroplasty.
Glucosamine sulfate has a good tolerability profile and high safety. In all study protocols and meta-analyses, there were no statistical or clinically significant differences in the number and severity of adverse events compared with placebo. At the same time, comparative studies have shown the prevalence of adverse events when taking NSAIDs than glucosamine. A meta-analysis of randomized controlled trials showed that the most common gastrointestinal side effects, which, as a rule, are mild. Cancellation of treatment due to intolerance to the drug occurred in isolated cases. Cardiovascular events were observed in elderly patients, but they also occurred no more frequently than in those receiving placebo. Glucosamine sulfate did not increase insulin resistance.
Another structural analog of cartilage, chondroitin sulfate, also belongs to the symptomatic drugs of delayed action. It is a sulfated mucopolysaccharide and is part of the proteoglycan complexes that are synthesized by chondrocytes. For the full function of cartilage tissue, 2 conditions must be met: 1) a sufficient number of chondrocytes and 2) they must be metabolically active and synthesize a sufficient amount of extracellular matrix. The composition of the matrix includes chondroitin sulfate. Due to the presence of carboxyl and sulfate groups, glycosaminoglycans and, in particular, chondroitin sulfate, have a pronounced hydrophobicity, and this, in turn, contributes to the normal functioning of the cartilage and the preservation of its elastic properties. When taken orally, it is determined in high concentrations in the synovial fluid. Its biological activity is in many respects close to glycosamine.
The level of evidence for chondroitin sulfate for symptom modification in OA is as high as for glucosamine sulfate (IA), as reflected in the 2003 EULAR guidelines. Leed B.F. et al. conducted a meta-analysis of 7 controlled clinical trials, which involved 703 patients with damage to large joints (knee and hip), while 372 patients were treated with chondroitin sulfate and 331 were taking placebo. The duration of therapy ranged from 3 to 12 months, and the dose of the drug ranged from 800 to 2000 mg/day. The effectiveness of chondroitin sulfate was significantly higher compared with placebo in terms of such indicators as pain according to the VAS, the Lequesne index and the global assessment of patient outcomes. This review also analyzed the tolerability of the drug, which was good and was comparable with placebo. Adverse events included abdominal pain (in 18 out of 349 patients), diarrhea (in 7), constipation (in 2), skin symptoms (in 4), eyelid edema (in 1), lower extremity edema (in 1 ), alopecia (in 1) and extrasystole (in 1).
Uebelhart D. et al. evaluated the efficacy and tolerability of two courses of oral chondroitin sulfate therapy lasting 3 months for 1 year in a randomized, double-blind, multicenter, placebo-controlled study in 120 patients with symptomatic OA of the knee. Primary efficacy was considered by assessing the Lequesne algo-functional index, and secondary - by the dynamics of VAS, the speed of passing a certain distance, the global assessment of the effectiveness of therapy, and the need for paracetamol. The width of the joint space was assessed in the medial part of the tibiofemoral joint. The intent-to-treat analysis included 110 out of 120 patients. By the end of the observation, the algo-functional index decreased by 36% in the main group and by 23% in the control group. Further analysis showed that chondroitin sulfate had not only a significant symptom-modifying, but also a structural-modifying effect. By the end of the year, there was a further decrease in the joint space in patients who took placebo, which was not recorded during chondroitin therapy.
The chondroprotective effect of chondroitin sulfate is also expressed in the so-called aftereffect of the drug, i.e. continued improvement in OA symptoms after discontinuation of treatment with this drug. The author emphasizes that the structure-modifying effect of this drug has been proven both in experimental and clinical studies, and a positive property of chondroitin is its low toxicity even with prolonged use.
Combined preparations with chondroprotective activity include artra, kondronova and Teraflex. Teraflex (Bayer, Germany) includes 500 mg of glucosamine hydrochloride and 400 mg of sodium chondroitin sulfate. It is prescribed 2 capsules 2 times a day for the first 3-4 weeks, and then 2 capsules a day. Duration of admission, as a rule, is 6 months.
The therapeutic activity of Teraflex has been proven in several clinical studies. In an open study conducted at the Institute of Rheumatology of the Russian Academy of Medical Sciences, L.I. Benevolenskaya et al. studied the efficacy, tolerability and safety of Teraflex in 50 patients with gonorrhea and coxarthrosis. All patients had clinically pronounced osteoarthritis with pain, morning stiffness and functional insufficiency of the musculoskeletal system, as well as the need to take NSAIDs. The duration of observation was 6 months, and in the first 4 months, patients took 2 capsules of Tera-flex together with 1200 mg of ibuprofen. When a positive effect was obtained, it was possible to reduce the daily requirement for ibuprofen up to its complete abolition. By the end of 4 months of continuous therapy, Teraflex led to a significant decrease in the total WOMAC index, while there was a significant positive dynamics in the intensity of joint pain, morning stiffness, and functional insufficiency of the affected joints. In 26 out of 50 patients, the daily requirement for ibuprofen was reduced. According to patients, improvement by the end of the second month. therapy was observed in 77.8% of cases and by the end of the fourth - in 74.4%, and according to the doctor - in 88.6 and 83.7%, respectively. Interestingly, in the next 2 months after stopping treatment, the therapeutic efficacy of Teraflex continued to be maintained. In this study, the drug was well tolerated. Adverse events were observed only in 6 patients and were mainly associated with ibuprofen. Teraflex in isolated cases caused pain in the upper abdomen and stool retention.
Another 6-month open randomized multicenter trial also evaluated the effectiveness of Teraflex in patients with clinically significant knee osteoarthritis and spondylosis deformans. In all patients, pain during walking on the VAS scale was above 40 mm, and the radiographic stage corresponded to stages I-III according to Kellgren and Lawrence. Patients of the first (main) group took Teraflex with diclofenac and the second (control) - only diclofenac. By the end of 3 months in the main group, the intensity of joint pain significantly decreased and remained at this level until the end of 6 months. treatment. In the second group, a positive trend in this indicator was also observed, although to a lesser extent compared to the main group. A similar trend was noted in the WOMAC functional index. By the end of 6 months treatment in the first group, according to the doctor, a significant improvement was registered in 23.3% of patients and an improvement in 60%, and in the control group - in 16.7 and 40%, respectively. At the same time, the ineffectiveness of the therapy was registered in 23% of patients taking diclofenac, and only 3.3% in the group of patients who, along with diclofenac, took Teraflex. As in the previous study, Teraflex was well tolerated. In total, 5 adverse events were detected in the main group and 8 in the control group. While taking Teraflex, heartburn, pain in the upper abdomen, and flatulence were observed, which were mild and did not require discontinuation of treatment with this drug. In one case, an allergic reaction was observed, accompanied by a skin rash.
Of great interest is the multicenter, double-blind, Glucosamin/|chondroitin Arthritis Intervention Trial (GAIT), conducted in the United States under the auspices of the National Institutes of Health. This study included 1583 patients with symptomatic OA of the knee. All patients were divided into 5 groups. In separate groups, patients received either 1500 mg of glucosamine hydrochloride, or 1200 mg of chondroitin sulfate, or a combination of glucosamine and chondroitin, or 200 mg of celecoxib or placebo. The duration of therapy was 24 weeks. The primary point was a decrease in the intensity of pain on the WOMAC scale in the knee joints by 20% or more by week 24. Despite the controversial design of this study and the large percentage of patients who experienced a significant reduction in pain intensity on placebo, interesting data were obtained. Patients with initially severe or severe pain in the knee joints were the most in the group on combined therapy with glucosamine and chondroitin and statistically significantly higher compared to the placebo group (79 and 54.3%, respectively, p = 0.002). Adverse events were rare, were moderately severe and occurred approximately equally often in separate groups.
The expediency of combination therapy and its structure-modifying effect is confirmed by experimental data on the study of the effectiveness of the simultaneous use of chondroitin sulfate and glucosamine hydrochloride. Combination therapy on the OA model in rabbits contributed to an increase in the production of glycosaminoglycans by chondrocytes by 96.6%, and against the background of monotherapy with structural analogs of cartilage - only by 32%. With combination therapy, cartilage damage was also less severe compared with the use of glycosamine or chondroitin. It should be borne in mind that the structural analogues of cartilage have not only common, but also distinctive mechanisms of their effect on pain and inflammation. At the same time, they are synergists and, when used together, complement and enhance each other's action.
Thus, Teraflex has a clear symptom-modifying effect in patients with OA, which is manifested by a decrease in the intensity of pain and an improvement in the function of the affected joints. It also reduces the daily requirement for NSAIDs. As for the evidence of its structural-modifying properties, this requires long-term treatment with this drug (for a number of months or even years) with a thorough analysis of the width of the joint space according to X-ray and MRI studies, as well as determining the volume of articular cartilage before and after conducting such therapy.
At present, the question of the direct chondroprotective action of structural analogs of cartilage is being resolved ambiguously. More and more researchers adhere to the point of view that the so-called chondroprotective drugs not only stimulate the synthesis of the cartilage matrix, i.e. proteoglycans, glycosaminoglycans and hyaluronic acid by chondrocytes, how much they have an anti-inflammatory effect, which is realized with their long-term administration. A positive solution to this problem is largely due to the lack of highly informative methods that allow one to adequately judge the safety of cartilage tissue and meet the requirements for the criteria for the progression of OA. In this regard, it seems relevant to distinguish between clinical criteria for OA and factors not associated with this disease, to identify features of the progression of OA of the hip joint, different from OA of the knee joint. No less relevant is the conduct of further in-depth studies in this disease to clarify the relationship between clinical, radiological, arthrosonographic and MRI data.

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