PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART II. ANTIMICROBIAL AGENTS

CHAPTER 11. THE USE OF MICROORGANISMS IN DRUG MANUFACTURING AND LABORATORY RESEARCH

Microorganisms and their metabolic products are widely utilized for the Diagnosis, Treatment, and Prevention of diseases. Alongside Vaccines, immune sera, and Antibiotics, numerous other preparations have long been established in medical practice. Novel BIOLOGICALLY ACTIVE SUBSTANCES are currently produced and developed using recombinant microbial strains engineered via genetic technologies (Chapters 8 and 9). Microorganisms also serve analytical purposes in evaluating the mutagenic potential of chemical compounds, Vitamins, Amino Acids, and related substances, as well as acting as models to test The impact of certain pharmaceutical products on METABOLISM.

11.1 Medicinal Products Produced by Microorganisms

Dextrans are synthesized by lactic acid Bacteria of the genus Leuconostoc (L. dextranicum and L. mesenteroides) when grown on sucrose as a carbon source. These polymers consist of glucose residues linked by α-1,6-bonds, with a variable molecular weight ranging from 15,000 to 20,000,000 Da (Fig. 64).

Class="center">Fig. 64. Structure of dextran.

Dextrans of a specific molecular weight can be tailored for medical Applications. They are obtained either through the acid Hydrolysis of high-polymer dextran or by introducing low-molecular-weight dextrans as precursors into the culture medium. These act as polymerization centers, enabling the yield of low-molecular-weight dextran.

Dextrans are manufactured on an industrial scale and utilized as plasma expanders. They can be administered intravenously or applied topically in the treatment of ulcers and Burns, in which case they form a hydrophilic film that absorbs exudates. An iron(III) hydroxide-dextran complex with a Molecular Weight of 5,000–7,000 Da is administered intravenously to treat iron-deficiency anemia when oral therapy is ineffective or contraindicated. The sodium salt of dextran sulfuric acid esters (dextran sodium sulfate) exhibits anticoagulant properties similar to heparin and is administered intravenously [11, 13].

As plasma substitutes, dextrans must possess a molecular weight between 40,000 and 300,000 Da. Polymers with lower molecular weights are rapidly cleared from the body, whereas those with higher molecular weights pose potential risks due to potential accumulation in Tissues. In clinical practice, infusion solutions with average molecular weights of 40,000, 70,000, and 110,000 Da are commercially available.

These solutions contain 6–10% dextran, 0.9% sodium chloride, and 5% glucose in Water. They are sterilized by autoclaving and tested for pyrogenicity, toxicity, and sterility.

Dextrans serve as the basis for manufacturing Sephadex gels, which are widely employed in chromatographic applications.

11.2 Vitamins, Amino Acids, and Organic Acids

These compounds, produced by microorganisms and utilized in pharmaceutical manufacturing, are summarized in Table 22. They are obtained through biotechnological Methods, the ongoing advancement of which continually expands this range of products and improves their clinical accessibility.

Table 22. Selected Bioactive Substances Produced by Microorganisms

11.3 Iron-Chelating Agents

When grown in iron-deficient media, numerous microorganisms synthesize iron-binding compounds—typically phenolates or hydroxamates—known as siderophores. A prolific producer of the siderophore desferrioxamine (Fig. 65) is Streptomyces pilosus.

Fig. 65. Structure of desferrioxamine and its chelate complex.

Desferrioxamine (desferal) is a potent antidote utilized in acute iron poisoning. The latter may occur, for instance, in children who ingest iron sulfate for uncomplicated iron deficiency. Desferal exhibits high affinity for iron (with a binding constant of approximately 10³⁰); its metal complex is water-soluble and readily excreted from the body. In hemolytic anemias such as thalassemia, desferal is administered alongside Blood transfusions to maintain normal iron and Hemoglobin levels. Desferal is available as a sterile powder and is administered via injection, or orally in cases of acute iron poisoning to clear the gastrointestinal tract.

Siderophores hold significant therapeutic potential in the treatment of malignant and viral diseases. Paracoccus denitrificans produces siderophores that inhibit tumor Cell proliferation and viral RNA Replication. This activity is attributed to the induction of iron starvation, an element essential for these metabolic processes.

11.4 Enzymes

Microorganisms produce a variety of enzymes employed as therapeutic agents or for diagnostic purposes. The latter will be discussed below.

Streptokinase, produced by streptococci, is capable of transforming plasminogen into plasmin, a protease that causes the dissolution of blood clots. It is used in the treatment of deep vein thrombosis, acute arterial thrombosis, and acute Pulmonary Embolism. This enzyme may also be used in cases of myocardial infarction.

Streptodornase, also produced by streptococci, helps liquefy pus. It is a deoxyribonuclease that hydrolyzes deoxyribonucleoprotein and DNA, which are responsible for the viscosity of pus. The combined use of streptokinase and streptodornase is effective in treating chest cavity diseases accompanied by The formation of blood clots and pus, as well as in the treatment of purulent wounds.

Both enzymes are obtained by cultivating non-pathogenic streptococcal strains in a medium with an excess of glucose. The enzymes are isolated from the culture liquid and produced in injectable form.

L-asparaginase is produced by E. coli and Erwinia carotovora. The enzyme is used in the Chemotherapy of certain forms of leukemia. L-asparaginase cleaves one amino group from asparagine, converting it into aspartic acid. The selectivity of the enzyme's action is determined by the requirement of certain tumor cell types for asparagine, whereas normal Cells do not require it.

Neuraminidase is obtained by cultivating Vibrio cholerae. The enzyme cleaves N-acetylneuraminic acid residues present in the membrane of certain tumor cells, thereby increasing their antigenic activity. It can be used in the treatment of certain forms of leukemia.

β-lactamases, which inactivate Penicillins and Cephalosporins, are used to determine the sterility of these antibiotics (see below) or in the MICROBIOLOGICAL ANALYSIS OF clinical material from patients receiving these antibiotics. For therapeutic purposes, they are used in cases of severe allergic reactions to β-lactam antibiotics. The enzyme is administered intramuscularly or intravenously along with other drugs (adrenaline, antihistamines).

11.5 Microbial Biotransformation of Drugs.

11.5.1 Microbial Biotransformation

The enzyme systems of microorganisms make it possible to carry out chemical conversions of biologically active substances to obtain pharmaceutical preparations. Such reactions under chemical synthesis conditions usually take place at harsh temperatures and pH levels and are multi-stage processes. Microbial transformation is carried out under physiological conditions, which helps preserve product activity and usually proceeds in a single stage.

The advantages of microbial transformation were first discovered during The production of steroid drugs. One of the essential stages of this process is the hydroxylation of the precursor molecule at a specific position. Many microorganisms possess this capability. Fig. 66 shows an example of biological transformation involving Rhizopus nigricans. Immobilized cells are used for the biotransformation of Steroids and antibiotics, as well as for obtaining the antiviral drug adenin arabinoside. The process involves transglycosylation.

Fig. 66. Transformation of progesterone by the enzyme system of Rhizopus nigricans

Microorganism strains have been found that are capable of converting hydrocortisone and cortisone into prednisolone and prednisone, respectively, via dehydrogenation, etc.

Biotransformation processes can utilize cells immobilized in a polymer gel matrix, as well as membrane systems that allow the process to be carried out continuously and simplify product purification. Immobilized cells are used for the biotransformation of steroids and antibiotics, and for producing the antiviral drug adenin arabinoside. The production process of the latter substance involves a transglycosylation reaction (uracil arabinoside + adenine ^ adenin arabinoside) carried out by Enterobacter aerogenes cells immobilized in polyurethane.

In antibiotic production, an essential step in obtaining certain penicillins is the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid using microbial penicillin acylase. In this case, good results are achieved by using recombinant strains with high enzymatic activity. Cell immobilization increases their stability and enhances product yield.

11.5.2 Insecticides

Entomopathogenic bacteria, Viruses, and Fungi can be used to control insect pests. Biological pest control methods are environmentally safer than chemical control methods. However, microorganisms proposed for this purpose are subject to strict safety requirements regarding mammals and plants.

The toxins of Bacillus thuringiensis have been studied the most. The δ-toxin is a protein contained in spores in the form of crystalline inclusions, active against Lepidoptera larvae (moths, butterflies). Activation of the toxin occurs upon its limited hydrolysis in the larval gut, after which the gut walls are destroyed, resulting in the insect's death. The insecticidal preparation is produced in the form of a powder containing spores and toxin crystals. It is non-toxic to humans and animals and is used to protect crops from caterpillars. A new strain of B. thuringiensis has been found whose δ-toxin has a broader spectrum of activity and is more active against Coleoptera (beetles) than against Lepidoptera and Diptera (flies and mosquitoes).

The second toxin of B. thuringiensis, the β-toxin, acts on all the aforementioned insect families; it is an adenine nucleotide, possibly an analogue of ATP, which competitively inhibits enzymes involved in ATP hydrolysis. It is toxic to mammals, which is why the insecticide production utilizes a B. thuringiensis strain that produces only the δ-toxin.

11.6 Application of Microorganisms and Their Products in Laboratory Research

11.6.1 Determination of Vitamins and Amino Acids

Auxotrophic strains of microorganisms are used as test cultures, i.e., those for which the substance being determined is an essential growth factor. The nutrient medium must contain all substances necessary for the test Organism, aside from the one being determined. The latter is introduced into wells made in the Agar medium in Petri dishes. Within certain limits, the diameter of the culture growth zone around the well will be proportional to the concentration of the growth factor, which is determined using a standard curve. This method is mainly used in vitamin analysis, as Amino acids are usually determined by chemical methods.

11.6.2 Phenylketonuria

This is an inherited metabolic disorder in which the body is unable to convert excess phenylalanine (PA) into Tyrosine, which is required for The Biosynthesis of thyroxine, adrenaline, and noradrenaline. Diagnostic signs of the disease include the presence of phenylpyruvic acid (PPA) in the urine and elevated levels of PA and PPA in the blood. Patients are prescribed a low-PA diet. Without taking necessary measures, mental retardation develops. To diagnose phenylketonuria, it has been proposed to use Bacillus subtilis, whose growth on a minimal medium is inhibited by β-2-thienylalanine (thienyl), but restored in the presence of PA or PPA. Test blood or urine samples are applied to filter paper discs, which are placed on the inoculated medium in Petri dishes. In a positive result, growth Zones of the microorganism are observed around the discs. The diameter of the zones is proportional to the content of the substances being investigated, the quantity of which is determined using a standard curve.

11.6.3 Determination of Carcinogens and Mutagens

All chemotherapeutic agents must undergo screening for mutagenic and potentially carcinogenic activity. A rapid method using mutant strains of Salmonella typhimurium has been proposed for this purpose. These strains carry Mutations in the histidine Operon and are unable to synthesize histidine. Two additional mutations increase the sensitivity of the test system: the first causes a structural defect in the lipopolysaccharide of The Cell wall, thereby increasing its permeability to large hydrophobic molecules, while the second mutation disrupts the DNA Repair system, preventing the restoration of Introduction/20.html">DNA Structure following exposure to a mutagen.

The test culture is mixed with the test substance in a molten (45°C) minimal agar medium and poured into a Petri dish. An overlay of agar medium containing trace amounts of histidine is applied on top, allowing the cells to undergo a few division cycles, which is necessary to detect the Action of Certain mutagens. Following a 48-hour incubation at 37°C, revertant colonies are counted and compared with those on a control plate lacking the mutagen. A revertant colony is the progeny of a cell that has mutated back to the wild type and regained The ability to synthesize histidine (to grow on minimal medium).

11.6.4 The Use of Microbial Enzymes in Sterility Testing

When assessing sterilization efficacy, spore-forming microorganisms are utilized as part of test systems. In sterility testing of antibiotic preparations, microbial enzymes that inactivate these substances are employed. Membrane filtration, also used for this purpose, has such drawbacks as the risk of accidental microbial contamination of the filters and the adsorption of the antibiotic onto the filter, with its subsequent release into the nutrient medium. The use of inactivating enzymes is a more reliable approach. Appropriate β-lactamases have been proposed for the inactivation of penicillins and cephalosporins. Chloramphenicol can be inactivated by acetyltransferase, and Aminoglycosides through enzymatic phosphorylation, Acetylation, or adenylylation. The Pharmacopeia recommends using only β-lactamases, while the other methods have not yet found widespread application.

11.6.5 Application of Immobilized Enzymes

The USE OF IMMOBILIZED enzymes holds great promise for developing test systems for clinical Diagnostics. For instance, glucose oxidase, used in blood glucose determination, is obtained from the cultivation of Aspergillus niger. A method has been proposed for immobilizing this enzyme onto a platinum electrode, which can measure oxygen consumption during glucose oxidation. However, this system has several limitations that must be addressed before clinical Implementation.

11.6.6 Microorganisms as Model Systems in Drug Metabolism Studies

Pharmaceutical agents undergo rigorous efficacy and toxicity testing, which includes investigating their Metabolic Pathways in mammals, such as potential chemical transformations, distribution in Organs and tissues, and clearance rates. These tests are primarily conducted on animals, with microsomal systems, tissue cultures, and perfused organs used to a lesser extent. Microbial cells have been proposed as an in vitro model of metabolism due to the similarities between certain enzyme systems in MICROORGANISMS AND THE human Liver. A major advantage of such models is their relatively low cost and the ability to obtain metabolites in quantities sufficient for analysis. To develop these model systems, A large number of microorganisms are screened for their ability to metabolize a given substance. The selected strain is grown in shake flasks containing the test substance, and samples are taken at regular intervals to detect drug metabolites. If necessary, the process is scaled up to yield larger quantities of metabolites for Structural and biological activity studies.