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.
Planner
The principle of chemotherapy
Determination of antibiotics
Classification of antibiotics. Chemical classification.
Obtaining methods:
а) microbial synthesis;
b) chemical synthesis;
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)
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).
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.)
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.
Antibiosis is 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.
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.
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
Classification of antibiotics
Main and reserved
By the types of producer
According to the biological action type
According to the chemical structure.
Classification of antibiotics according to the chemical
structure
1. 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) and
etc.; 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.
14-members macrolides (erythromycin, oleandomycin,
claritromycin, roxitromycin and other)
15- members azalides (azitromycin);
16- members macrolides (spiramycin and other)
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)
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.
6. Antibiotics of other structure
Lincosamides (lincomycin, clindamycin);
Ansamycin (rifampicin) – antibiotics contain the aromatic
nucleus in their molecules (usually
naphthalene), 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.
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 βlactams, 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.
Chemical classification of antibiotics
1)
2)
3)
4)
5)
6)
7)
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
We can classify antibiotics by their mode of action.
Antibiotics that influence on Gram +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 on Gram -ve cell wall of
bacteriums (polymyxin violate 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.
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).
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:
1). Modified method of a diffusion in an agar
2). Ureased
3). Fermentative
4). Radioimmunoassay
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).
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.
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.
Toxicity investigation of antibiotics
1.
2.
3.
4.
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 1020 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.
1.
2.
3.
4.
SPhU (anomal toxicity)
Quantity of mice - 5
Mass of mice - 17-22 г
injection – intravenous. Solution is
injected from 15sec till 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.
Investigation of the antibiotic’s pyrogenicity
1.
2.
3.
4.
Х Pharmacopoeia
Quantity of rabbits - 3
Mass of rabbits – 1,5-2,5 kg
injection– in ear vein, quantity of
solution is 10 ml on 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
1. Quantity of rabbits - 3
2. Mass of rabbits not less than 1,5 kg
3. injection - in ear vein, Injection
time is 4 minutes, injection volume
is 0,5 ml-10 ml on 1kg of mass
4. The investigation is realized on 3
rabbits. The sample keeps up the
investigation, if the total increasing
in temperature does not exceed than
1,15С, if the temperature rising is
1,15С, but not more than 2,65С.
The investigation is repeated. Then
the corresponding values are 2,8С;
and 4,3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 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.
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.
Acyclic antibiotics and their semi-synthetic
analogs (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 semisynthetic tetracyclines analogs.
Natural tetracyclines
Tetracycline (Tetracyclinum)
H3C
H3C
7
N
OH
5
6
CH3
4
8
5a
4a
3
OH
9
11a
12a
2
C
10
11
12
1
OH
NH2
O
OH
O
OH
O
(4S,4aS,5aS,6S,12aS)-4-(Dimethylamino)-3,6,10,12,12apentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide.
In medical practice tetracycline is used in the form of a base or
Natural tetracyclines: tetracycline, tetracycline h/ch,
oxytetracycline dihydrate, oxytetracycline h/ch,
Chlor(o)tetracycline h/ch (biomicyn)
Chlor(o)tetracycline
Oxytetracycline
(terramicyn)
Tetracycline
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.
Identification
1. The reaction the a concentrated sulfuric acid – forming
the anhydrotetracycline’s derivatives, which have a specific
colour (tetracycline h/ch – violet-red, oxytetracycline h/ch –
red), the colour transfers in a yellow at the adding of water:
2.
3.
4.
5.
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.
6.
7.
8.
9.
Formation of the coloured complex salts in an alcohol medium
with FeCl3- brown and red-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.
Formation of the azo dye dues to the present of phenolic
hydroxyl:
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.
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
Application
Broad-spectrum antibiotics at 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.
oxitetramicyn h/ch is included in the ointments
composition “Hyoxyson”, “Оxycort”, aerosol
“Oxycyclosol”.
Semi-synthetic tetracyclines
Methacycline h/ch
Rondomycin
Generic name (Methacyclini hydrochloridum)
H3C
CH2
OH
CH3
N
OH
C
OH
OH
O
OH
*HCl
NH2
O
O
4-Dimethylamino-1,4,4а,5,5а,6,11,12а-octahydro-3,5,10,12,12аpentaoxy-6-methylen-1,11 –dioxo-naphthacene-2-carboxamide
hydrochloride
Doxycycline hyclate (Doxycyclini hyclas) (SPhU),
Doxybene, Doxycycline hydrochloride
H3C
CH3
OH
CH3
N
OH
C
OH
OH
O
OH
* HCl * 1/2 C2H5OH * 1/2 H2O
NH2
O
O
H/cl hemiethanol hemi-hydrate (4S,4aR,5S,5aR,6R,12aS)4-(dimethylamino)-3,5,10,12,12а-pentahydroxy-6-methyl1,11-dioxo-1,4,4а,5,5а,6,11,12а-octahydrotetracene-2carboxamide
Doxcycline monohydrite - Vibramycin, Doxy-М, Unidox solutab
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:
1. TLC-method.
2. With a concentrated sulfuric acid appearance yellow
colour.
3. It gives reaction of chloride.
Quantitative determination.
1. Liquid chromatography.
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 accumulate in 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.
Thanx for attention