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  1. Lecture №4 General characteristic of antibiotics, their types. The main stages of the industrial production of antibiotics and the creation of new drugs in this series. The identification methods, the purity tests and quantitative determination of antibiotics. Using in medical practice, their side effects. Antibiotics of the alicyclic series. ass. Kozachok S.S.

  2. Planner • The principle of chemotherapy • Determination of antibiotics • Classification of antibiotics. Chemical classification. • Obtaining methods: а) microbial synthesis; b) chemicalsynthesis; c) Combining of the microbial and chemical synthesis • Analysis methods of antibiotics. • Determination the antibiotics units of action • The side effects of antibiotics. • Antibiotics of alicyclic structure(tetracycline)

  3. Chemotherapeutic agents –drugs wich have a selective activity against to certain infectious and parasitic diseases (sulphonylamides, antibiotics, antibacterial agents of the various chemical structure, antitubercular, antisyphilis, antiviral, antiparasitic, antihelminthic).

  4. Depending on the source and method of the producing of chemotherapeutic agents can be divided into 2 groups:Antibiotics - substances of natural origin, or the substaces are obtained by the modifying of natural substances;Original definition of antibiotics was material derived from microorganisms that suppress growth and sometimes kill the microorganism. Synthetic material was added later, when they were able to make synthetic antibiotics.Synthetic chemotherapeutic agents (sulfanilamide, nitrofurans, quinolones, nitroimidazoles, etc.)

  5. The principle of chemotherapy • Selectivity of the drug’s action against to certain microorganisms (the certain spectrum of the antimicrobial action) • Low toxicity to humans and animals. Antibiosisis the base of antibiotic’s action. Antibiosis is the phenomenon of the antagonism of microorganisms, the first was opened by Louis Pasteur in the 80 years of the XIX century.         For the first time the term "antibiotic" was offered by SA Waxman (USA). The word "antibiotics" comes from the Greek anti ("against") and bios ("life"). Antibiotics are drugs that either destroy bacteria or prevent their reproduction.

  6. Antibiotics– chemotherapeutic agents of a biological origin that have the ability to selectively kill or inhibit the growth of pathogenic agent (bacteria, viruses, fungi, protozoa microorganisms) or delay the development of malignant tumor.Antibiotics are used in medical practice, are produced by actinomycetes (radiant mushrooms), fungis and some bacterias.

  7. Antibiotics that are obtained from a plant origin called phytoncyds. Antibiotics are the representatives of the secondary metabolites of living cells. Primary metabolites are called the substance, that are necessary for cell growth (amino acids, carbohydrates, nucleotides, vitamins, etc.). Secondary metabolites are low molecular weight compounds (antibiotics, alkaloids, pigments ments, etc.) produced in the cells after the growing phase. Antibiotics have the following functions in the living producing cells:1. Means of attack and defense ("chemical weapons" cells);2. Detoxification from the harmful metabolites;control of some reactions in metabolism;3. Participation in the process of cell differentiation;4. Reserve nutrients

  8. Classification of antibiotics • Mainandreserved • By the types of producer • According to the biological action type • According to the chemical structure.

  9. Classification of antibiotics according to the chemical structure • β-lactams • Antibiotics, which are present in the molecule β- lactam cycle. • bicyclic: derivatives of 6-aminopenicillic- acid (penicillins), derivatives of 7 - aminocephalosporinic acid (cephalosporines) andetc.; monocyclic: monobactams and others; • 2. Macrolides and azalides are the • antibiotics, which contain a lactone ring with 14 - 16 atoms in their molecule. In azalides (azitromycin) the atom of nitrogen is present in a cycle.

  10. 14-members macrolides (erythromycin, oleandomycin, claritromycin, roxitromycin and other) 15- members azalides (azitromycin); 16- members macrolides (spiramycin and other)

  11. 3. Aminoglycosides Antibiotics which have the cyclohexan structure with OH- and NH2- or guanidino-derivatives and glycoside derivatives with one or certain OH-groups. Derivatives of D-streptydine (streptomycin); Derivatives of 2-desoxy-D-streptamine (Kanamycin, gentamicin, amikacin sulfate (amikin) and others)

  12. 4. Tetracyclines Antibiotics, which have the partially hydrated nucleus of tetracene. Tetracyclines, oxytetracyclines, doxycyclines and others. 5. Amphenicoles Chloramphenicol (levomycetin) have practical using, which according to its structure belongs to nitrophenylalkylamines.

  13. 6. Antibiotics of other structure Lincosamides (lincomycin, clindamycin); Ansamycin (rifampicin) – antibiotics contain the aromatic nucleus in their molecules (usuallynaphthalene), which in two positions contains an aliphatic chain, consisting of 15 - 20 carbon’s atoms Anthracycline(rubomycin, doxorubomycin, karminomycin hydrochloride (carubicin))– glycosides in which the aglycone is a substituted tetrahydronaphtacenquinon; Polyenes antibiotics(nystatin, levorin) – macrolides, which molecules contain a system of conjugated double bonds; Glycopeptide (vancomycin) and other.

  14. Bactericidal drugs kill the target bacterium or fungus and are more effective. You would use these types of drugs to treat endocarditis, meningitis, osteomyelitis, and other invasive bacterial infections. Hosts with low immune systems should also be treated with bactericidal drugs. These drugs include β- lactams, aminoglycosides, and fluoroquinolones. Bacteriostatic drugs inhibit growth and can be helpful cuz they let the normal host defenses to destroy the microorganisms. You would use these to treat infections such as cystitis. These drugs include tetracycline, sulfonamides, clindamycin, and chloramphenicol. Some organisms require 2 agents for killing such as entercocci.

  15. Chemical classificationof antibiotics • Acyclic structure (tetracycline) • Aromatic structure (levomicetine and its derivatives). • Heterocyclic structure (penicillins, cephalosporins). • Glycosides:        a) streptomycin;        b) the aminoglycosides (canamycin, neomycine, gentamicine, monomycin);        c) macrolides (erythromycin, oleandomitsine);        d) anzamitsiny (rifampicin). • Polyenes (nystatin, amphotericin). • Polypeptide structures (gramicidin, polymyxin) • Antineoplasmic: a) aureolic acid derivatives; b) anthracyclines; c) derivatives of quinoline-5,8-dion; d) actinomycin

  16. We can classify antibiotics by their mode of action. • Antibiotics that influence onGram +ve cell wall of bacteriums. Penicillins violate the synthesis of the bacterial cell wall - showing a bactericidal effect; macrolides violate the intracellular protein synthesis on the ribosome’s level - bacteriostatic action. • Antibiotics that influence onGram -ve cell wall of bacteriums (polymyxinviolate the penetration of the cytoplasm membrane (bactericidal effect). • Broad-spectrum antibiotics. Chloramphenicol and tetracycline (bacteriostatic effect on the level of ribosomes), cephalosporins (bactericidal action - inhibit synthesis of bacterial cell walls). Streptomycin, neomycin, kanamycin (bacteriostatic action - inhibit protein synthesis at the ribosome’s level). Rifampicin violate the synthesis of RNA.

  17. Methods of antibiotics obtaining • Microbiological synthesis is based on the mold (Penicillium) or the radiant mushrooms (Streptomyces). There are obtained by this method such antibiotics as: tetracyclines, the natural penicillins, glycosides, macrolides. The main stages of microbiological synthesis are:a) selection of a highly producing strains;b) selection the medium (pH, metal salts, carbohydrates, fats);c) the process (biosynthesis) of fermentation;d) isolation and purification of antibiotic (crystallization, chromatography, extraction, resedimentation).

  18. 1). Modified method of a diffusionin an agar2). Ureased3). Fermentative4). Radioimmunoassay • Chemical synthesis(levomycetin). • A combination of microbiological and chemical methods. Obtaining semi-synthetic antibiotics based on the transformation of the molecules of natural antibiotics (semisynthetic tetracyclines, penicillins, cephalosporins, etc.). Modern methods of determining the biological activity of antibiotics:

  19. Quantitative determination of antibiotics by a microbiological method Activity of antibiotics is determined by a comparing of the suppression growth degree of the test antibiotic on the sensitive (test cultures) m /o (microorganism) with a standard sample of antibiotic with known concentrations. Quantitative determination is carried out by the diffuse or turbidimetric methods. Calculation of the biological activity is performed according to a standard curve that was constructed on the basis of the definitions of the five concentrations of standard sample of the antibiotic. Biological activity is expressed in units of action (UA).

  20. U.A. – the minimum molar amount of the antibiotic, which inhibits the development of a test microorganism in a certain volume of the medium. The number of grams of active ingredient in U.A. is variety for different antibiotics! The average value of the activity, which was found by a biological method, somewhat lower than the theoretical activity. In the pharmacopoeial article is the theoretical value of the activity and the lower permissible boundary of the activities of the investigated antibiotic U.A. / mg. It was established that 1 mg of streptomycin equivalents to 1000 UA. Respectively, 1 UA of streptomycin equivalents to 1 microgram of a pure streptomycin basis. For other antibiotics UA differs from 1 microgram of the substance. For example, for benzylpenicillin 1 UA equivalents to = 0.6 mcg, that 1 mg of the antibiotic contains 1,667 UA.

  21. SIDE EFFECTS OF ANTIBIOTICS(according to Charkevych) • Sperinfection (all) • Allergy (all) • Dispepsia phenomenon, pain feeling • Local irritating action – at inner muscular injection; phlebitis, thrombophlebitis at intravenous injection • Effect on a liver (tetracycline) • Effect on a kidneys (cephalorydin, neomycin, polymyxin) • Hemodyscrasia (chloramphenicol) • Impairment of the function of vision and hearing (chloramphenicol in high doses) • Impaired function of a pair of VIII cranial nerve (aminoglycosides) • Mycosis (women - gynecologic diseases, male - a disease of the hearing aid) • Deafness, impaired tooth enamel, deposition in the bones - tetracyclines.

  22. Х Pharmacopoeia Quantity of mice - 5 Mass of mice – 18-20 g injection – intravenous , hypodermic, in an abdomen. Observation time according to the requirements specific pharmacopoeial monograph If, within the prescribed time there is no lost in mice, the medicine keeps up the investigation, in case of the death of a one or more mouse, the investigation repeat on the 10 mice with the weigh of 10-20 g. If during the secondary investigation , there is no lost in mice 10 mice, the drug holds the research . In the case of the death of a one mouse the medicine is defected. SPhU (anomal toxicity) Quantity of mice- 5 Mass of mice- 17-22 г injection – intravenous. Solution is injected from 15sectill 30sec, if there is no some remarks. The observation time is 24 hours, or a period of time according to the requirements of a separate article. Sample of the test is good, if none of the mice did not die within the specified time according to a separate article. In the case of the death of an animal test is repeated again. The sample test keeps up the investigation when the animals of the second group did not died. Toxicity investigation of antibiotics

  23. Х Pharmacopoeia Quantity of rabbits - 3 Mass of rabbits – 1,5-2,5 kg injection– in ear vein, quantity of solution is 10 mlon 1 kg of weigh. Injection time is 2 minutes. The solution is a pyrogen-free if after the injection no one of the rabbits do not have the rise in temperature more than 0,6С comparing with the initial temperature of the rabbits and in the amount does not exceed than 1,4С. If one or two rabbits, temperature increases more than 1,4 С, the investigation is repeated on 5 rabbits, the solution is pyrogen-free, if there is no more than 3 of 8 rebbits have the rise in temperature on 0,6  0C and the total amount of the rise in temperature does not exceed than 3,7С. SPhU Quantity of rabbits - 3 Mass of rabbits not less than 1,5 kg injection - in ear vein, Injection time is4minutes, injection volume is 0,5 ml-10 ml on 1kg of mass The investigation is realized on 3 rabbits. The sample keeps up the investigation, if the total increasing in temperature does not exceedthan 1,15С,if the temperature rising is1,15С, but not more than2,65С. The investigation is repeated. Then the corresponding valuesare2,8С; and4,3С. If the amount temperature rising is more than 4,3С. The sample is defected. If you need the research is conducted by another 3 or 6 rabbits.\ Investigation of the antibiotic’s pyrogenicity

  24. Investigation of antibiotics sterility To select the medium and test the suitability of the procedure’s methodic. This test is performed using the membrane filtration method or the method of a direct seeding. Regardless of the procedure’s method, realeze a corresponding negative control experiment using the samples, sterility of which has been proved earlier. The inoculation are periodically looked through during and after the incubation period, visually denote the presence of microorganism’s growth. If the test sample causes the turbidity of the medium, that makes impossible of the visual record, then after 14 days from the beginning of the incubation from each of the vessels there is carried a certain amount of the medium in the vessels with the same medium. Continue the incubation of the initial and repeating inoculations. The total incubation time is 14 + 7 days from the beginning of test. The medicine is sterile, if at the visual record tere is no microorganism growth.

  25. Investigation of the depressor substance This test is performed with the aim of exclusion the risk of blood pressure reducing (depressor reactions) after drug’s administration, if in medicine’s composition there was found the depressor substances (histamine, serotonin, bradykinin) at the prodaction. The investigation is realized on a cat, its mass is not more than 2 kg. The cat was narcotized by a chloralose, barbiturates or urethane to maintain a constant blood pressure.Through the carotid artery, the animal is attached to the instrument recording the blood pressure. In the femoral vein through the cannula is injected by a histamine and the test drug solutions. The test is an inefficacious if the reaction on 1.5 ml of histamine does not exceed the response to 1 ml. The sample fails the test if the average response to its injection is higher than the average responses to 1 ml of histamine per kg of a weight body, or if any one of its dose causes a greater depressor response than the final dose of a histamine solution.

  26. Acyclic antibiotics and their semi-syntheticanalogs (tetracyclines) Antibiotics, which have the partially hydrated nucleus of tetracene (naphthacene): This antibiotics are obtained from the microorganisms of Streptomyces aurofaciens i Streptomyces rimosus. In medical practice there is using the natural and semi- synthetic tetracyclines analogs.

  27. Natural tetracyclines Tetracycline (Tetracyclinum) (4S,4aS,5aS,6S,12aS)-4-(Dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide. In medical practice tetracycline is used in the form of a base or a hydrochloride(Tetracyclini hydrochloridum) (SPhU 1.2).

  28. Natural tetracyclines: tetracycline, tetracycline h/ch, oxytetracyclinedihydrate, oxytetracyclineh/ch, Chlor(o)tetracyclineh/ch (biomicyn) Chlor(o)tetracycline Oxytetracycline (terramicyn) Tetracycline

  29. Physical properties Tetracyclines are an yellow or an light-yellow crystalline powder, odorless, bitter on test. Their solutions in chloric acid rotate the polarized plane of a beam to the left. Soluble in water and their salts are a freely soluble in water. All substances of tetracyclines group are hard soluble in alcohol, but according to the amphoteric properties they are a freely soluble in diluted strong acids and bases. Basic properties are caused of the Dimethylamino group present in the 4-th position, and acidic - phenolic hydroxyl in 10-th position and the enol in 3-d and 12-th. The presence of in tetracycline molecule of two systems of conjugated double bonds at positions 1, 11 cause the characteristic absorption in the UV range.

  30. Identification 1. The reaction the a concentrated sulfuric acid – forming the anhydrotetracycline’s derivatives, which have a specific colour (tetracyclineh/ch – violet-red, oxytetracyclineh/ch – red), the colour transfers in a yellow at the adding of water:

  31. ТLC method compareing with a standard pharmacopoeial sample. • Hydrochloride’s of tetracyclines give the reaction of chlorides. • Determine an specific optical rotation. • UV-spectrometry. Determine the maximum and minimum absorption and calculate a specific absorption rate. • Formation of the coloured complex salts in an alcohol medium withFeCl3- brownandred-brown (present of the phenolic hydroxyl in the 10-th position). • Formation of the coloured complexes with the salts of copper (II), zinc. • Formation in certain conditions, the fluorescent products. • In an express analysis of tetracycline drugs there is used color reactions with sodium nitroprusside, n-dimethylaminobenzaldehide, Nessler reagent, diazoreagent.

  32. Formation of the azo dye dues to the present of phenolic hydroxyl:

  33. Quantitative determination • Liquid chromatography. Quantity is calculated in percent. • Tetracycline’s antibiotics are quantified by the biological method of diffusion in an agar. In this case, 1 mcg = 1 UA, then 1.0 g of material = 1000000 UA. • Spectrophotometry in the UV region, photocolorimetry and fluorimetry. Storage Protected from light.

  34. At the storage of tetracycline’s antibiotics, observing a change in color - darkening according to the formation of impurities of angidrotetratcycline and 4-epianhydrotratcycline and the products of their transformation. These substances are more toxic than the initial drugs. Aqueous solutions of the tetracyclines salts gradually becomes turbidity due to settle down a base. In weakly acidic medium tetracyclines hydrochlorid solutions are relatively stable, but in an acidic and a base medium, they are easily destroyed. For example, in an alkaline medium formed isotetracycline’s derivatives: tetracycline isotetracycline

  35. Application • Broad-spectrum antibioticsat pneumonia, dysentery, gonorrhea, typhoid and other infectious diseases. Administration inside in the form of tablets, capsules, suspensions, rarely inner muscular injections. Externally - in the form of ointments for burns treating, phlegmon and eye diseases. H.d. - 0,5 g; H.d.d. - 2,0 g. • Oletetrin – tetracycline + oleandomycin. • Vitacycline – tetracycline + vitamins С, В1, В2. • oxitetramicynh/chis included in the ointments composition “Hyoxyson”, “Оxycort”, aerosol “Oxycyclosol”.

  36. Methacyclineh/ch Rondomycin Generic name (Methacyclini hydrochloridum) 4-Dimethylamino-1,4,4а,5,5а,6,11,12а-octahydro-3,5,10,12,12а-pentaoxy-6-methylen-1,11 –dioxo-naphthacene-2-carboxamidehydrochloride Semi-synthetictetracyclines

  37. Doxycycline hyclate (Doxycyclini hyclas) (SPhU), Doxybene, Doxycycline hydrochloride H/clhemiethanolhemi-hydrate (4S,4aR,5S,5aR,6R,12aS)- 4-(dimethylamino)-3,5,10,12,12а-pentahydroxy-6-methyl-1,11-dioxo-1,4,4а,5,5а,6,11,12а-octahydrotetracene-2-carboxamide Doxcycline monohydrite - Vibramycin, Doxy-М, Unidox solutab

  38. Doxycycline hyclate Properties.Yellow, crystalline powder. Hygroscopic. A freely soluble in water, methanol, a slightly soluble in a 96 % ethanol alcohol, a practically insoluble in an ether. Identification: • TLC-method. • With a concentrated sulfuric acid appearance yellow colour. • It gives reaction of chloride.Quantitative determination. 1. Liquid chromatography.

  39. Apllication Doxycycline h/ch is highly effective for upper respiratory tract infection (bronchitis, pleurisy, pneumonia), has a prolonged effect (1-2 times take a day). Methacycline h/ch is better absorbed after oral administration (capsule), lasts longer in the blood and is effective in gonorrhea treating. Antibiotics of this group cause cross-resistance, but microorganisms get accustomed to tetracycline’s raw less than to penicillin. Tetracycline side effect Forming the complexes with Ca2+, Fe3+ions that’s why tetracyclines can accumulatein bones, tooth enamel. Do not recommend taking tetracyclines for children and pregnant women. When receiving these drugs should not eat dairy products, iron supplements and antacids medicines containing an aluminum, magnesium and calcium salts.

  40. Thanx for attention