Volume 02 Issue 12-2022
9
International Journal of Medical Sciences And Clinical Research
(ISSN
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2771-2265)
VOLUME
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12
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:
09-20
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893
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1121105677
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IF
–
5.654
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ABSTRACT
By chemical modification of polygalacturonic acid with anti-TB drugs isoniazid, ethambutol and rifampicin, the
macromolecular system Biomayrin was obtained. Pharmaco-toxicological studies have ascertained that Biomayrin is
less toxic than low-molecular counterparts and has pronounced anti-tuberculosis activity, and with a two-fold lower
dose when calculating the active substance, it has identical activity with the combined anti-tuberculosis drug Mayrin.
Pharmacokinetic studies have shown that with the introduction of Biomayrin, the therapeutic concentration of the
active ingredients (isoniazid, ethambutol and rifampicin) in the blood lasts longer than that of their low-molecular
counterparts. It should also be noted that the chemical binding of isoniazid to the polysaccharide matrix slows down
its metabolism to therapeutically inactive acetylisoniazid.
KEYWORDS
Polygalacturonic acid, isoniazid, ethambutol, rifampicin, mayrin, polymer- carrier, tuberculosis, prolongation.
Research Article
PRE-CLINICAL STUDIES OF THE ANTITUBERCULOUS DRUG BIOMAYRIN
Submission Date:
December 12, 2022,
Accepted Date:
December 17, 2022,
Published Date:
December 22, 2022
Crossref doi:
https://doi.org/10.37547/ijmscr/Volume02Issue12-03
Mamatmusaeva N.E.
Institute Of Bioorganic Chemistry, Academy Of Sciences Of Uzbekistan, Uzbekistan, Tashkent City,Mirzo
Ulugbek St. 83, Uzbekistan
Shomuratov Sh.А.
Institute Of Bioorganic Chemistry, Academy Of Sciences Of Uzbekistan, Uzbekistan, Tashkent City,Mirzo
Ulugbek St. 83, Uzbekistan
Sagdullaev B.Т.
Institute Of Bioorganic Chemistry, Academy Of Sciences Of Uzbekistan, Uzbekistan, Tashkent City,Mirzo
Ulugbek St. 83, Uzbekistan
Turaev А.S.
Institute Of Bioorganic Chemistry, Academy Of Sciences Of Uzbekistan, Uzbekistan, Tashkent City,Mirzo
Ulugbek St. 83, Uzbekistan
Journal
Website:
https://theusajournals.
com/index.php/ijmscr
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
Volume 02 Issue 12-2022
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(ISSN
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VOLUME
02
I
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09-20
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(2022:
5.
893
)
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–
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INTRODUCTION
In connection with the specificity of tuberculosis
treatment - complex therapy (simultaneously with
several anti-tuberculosis drugs), the duration of
treatment, the need to assign multiple doses of large
doses of medications and the occurrence of toxic-
allergic complications due to this - it is very important
to find a way to reduce the dosage of anti-TB drugs
[1,2] . One of the ways to improve the conditions of
drug therapy is the creation and use of polymeric anti-
tuberculosis drugs of prolonged action. Such
prolonged anti-TB drugs will not only increase the
duration of action of tuberculostatics, but also reduce
their side effects, reduce the overall consumption of
the drug. Such macromolecular drug systems can be
obtained by incorporating anti-tuberculosis drugs into
the macromolecules of the polymer carrier.
In this connection, we have synthesized the
macromolecular system by chemical binding of anti-
tuberculosis drugs: isoniazid, ethambutol and
rifampicin to a modified polygalacturonic acid
macromolecule (PGA) with a molecular mass of 16 kDa,
isolated from citrus pectin by demetoxylation. The
macromolecular system is called Biomayrin. In the
macromolecule of Biomayrin, isoniazid is chemically
bound to the macromolecule PGA through the
azomethine bond, ethambutol and rifampicin via the
ionic bond [3].
The chemical structure of Biomayrin can be
represented as polygalacturonic acid complexes with
4-pyridinecarboxylic acid hydrozone and [S-(R*, R*)] -
2,2 '- (1,2-ethanediyl diimino) bis (1-butanol) and
polygalacturonic acid complex with 3 - [[(4-methyl-1-
piperazinyl) imino] methyl] rifamycin:
(С6Н6О5)n(С6Н5N3O)m(C10H24N2O2)s+(C6H7)n(C43H58N4O12)h
n=20±3, m=13±3, s=10±3, h=5±2
O
COONH
2
CH
2
CH
2
NHCHCH
2
CH
3
HCl
CH
2
OH
CHCH
2
CH
3
CH
2
OH
+
.
H
H
H
O
N
CONHNH
n
O
NH
N
CH
N
N
O
CH
3
OH
OH
CH
3
OH
CH
3
OH
H
3
CO
CH
3
O
CH
3
O
H
3
C
HO
CH
3
O
O
CH
3
O
H
OH
OH
H
H
H
O
COO
m
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Biomayrin
This article presents the results of preclinical studies of Biomayrin.
METHOD
Objects of study: Anti-TB drugs:
Biomayrin: (isoniazid - 15 ± 3%, ethambutol - 15 ± 3%,
rifampicin - 17 ± 3%).
Rifampicin - Becton, Dickinson and Company Sparks.
Isoniazid - Becton, Dickinson and Company Sparks.
Ethambutol - Becton, Dickinson and Company Sparks.
Preparation of Biomayrin solutions:
Pure Substance: Biomayrin in Levenstein-Jensen
Nutrient medium
2. Final concentration = 4000 mg/l
dissolve 59.17 mg of Biomayrin substance in 5 ml of
ethanol, then add 10 ml of sterile distilled water
(heating of the solutions is possible);
2. Final concentration = 400 mg/l
11 ml of Solution I + 90 ml of sterile distilled water;
3. Final concentration = 200 mg/l
21 ml of Solution II + 20 ml of sterile distilled water;
4. Final concentration = 50 mg/l
10 ml of Solution III + 30 ml of sterile distilled water.
Table 1
The final concentration of the drug in the medium
The final concentration of the drug in the medium
The volume of medium (mg/l)
40.0
20.0
10.0
5.0
Distilled water (ml)
450
45
45
45
Solution I, ml
\
\
2.5
\
Solution II, ml
50
\
\
\
Solution III, ml
\
5.0
2.5
\
Solution IV
, в
ml
\
\
\
5.0
Volume 02 Issue 12-2022
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VOLUME
02
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12
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5.
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1121105677
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The study was conducted using the method of
absolute concentrations with the content of anti-TB
drugs on a nutrient medium Levenstein-Jensen in a
dose according to
McFarland №1
, the turbidity
standard is 10
6
-10
8
bacteria/ml. To determine the
susceptibility to anti-tuberculosis drugs, a virulent
Reference M. Tuberculosis H37Rv strain was selected.
To obtain reliable results, two dilutions of the bacterial
suspension were prepared from each culture of the
H37Rv strain:
Table 2
The composition of the statutory solution
Statutory solution
2 ml
К
1
0.9 ml NaCl = 0.1 statutory solution
or
1.8 ml of NaCl = 0.2 ml of the statutory solution
К
2
0.9 ml NaCl = 0.1 statutory solution
Then, two dilutions from each H37Rv strain were
inoculated into nutrient media containing anti-
tuberculosis drugs. Inoculated culture media were
incubated in a thermostat at 37°C for 28 days [4].
Bacteriostatic activity of Biomayrin in vivo was
performed on 20 ordinary guinea pigs weighing 350-
400 grams and 16 rabbits of the Chinchilla breed
weighing 2.5-3.0 kg with the virulent strains of
Mycobacterium tuberculosis “Humanis NQ 2520” [5 ].
Determination of acute toxicity. Acute toxicity and the
clinical picture of intoxication were determined on
white outbred mice (n=25) weighing 18-20 g of both
sexes. The drugs were administered in 1 and 3%
concentrations, depending on the dose once on an
empty stomach intraperitoneally. The effect of the
drugs was assessed by the change in behavioral
reactions, the general condition and death of animals
for 4 hours and within 14 days after drug
administration. The results of the experiments were
processed by the method of variation statistics. The
LD
50
calculations were made according to the method
of Litchfield and Wilcoxon [6].
Pharmacokinetics. Pharmacokinetic studies were
performed on outbred white male rats with an average
weight of 200±20 g. In studies based on the
composition of the active ingredients, the drug Mayrin
was chosen as a reference drug. Combined anti-TB
drug "Mayrin" where the main active ingredients are
isoniazid (75 mg), ethambutol (300 mg) and rifampicin
(150 mg), but in some combinations there is
pyrazinamide, which is produced under the names ACT-
4, Zukoks, Zukoks E, Zukoks Plus, Isozid comp 300 mg-
H,
Isocomb,
Isoprodian,
Kombitub,
Mayrin-P,
Prothiocomb, Lomicomb, etc. [7].
The concentration of isoniazid, its metabolite
acetylizoniazid and rifampicin was determined by high
Volume 02 Issue 12-2022
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VOLUME
02
I
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12
P
AGES
:
09-20
SJIF
I
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FACTOR
(2021:
5.
694
)
(2022:
5.
893
)
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–
1121105677
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–
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performance liquid chromatography on a Zorbax
Eclipse XDB C
18
column (3x100mm). Ethambutol was
determined spectrophotometrically [8]. Calculations
of pharmacokinetic parameters were performed using
the Borgia program [9].
RESULTS AND DISCUSSION
The results of drug susceptibility were assessed on the
28th day after sowing until pronounced growth in the
control tube. Grown strains in the control tube are
evaluated as sensitive if less than 20 colonies have
grown in vitro with abundant growth. Strains are
assessed as resistant if more than 20 colonies have
grown in vitro.
As can be seen from table 3, the strain H37Rv showed
sensitivity in all concentrations tested to Biomayrin.
Table 3
The sensitivity of the MBT (strain H37Rv) to the drug Biomayrin
The final
concentration of
drugs
H37Rv
Biomayrin
К
1
-10
3
К
2
-10
4
40.0
μ
g
negatively
negatively
20.0
μ
g
negatively
negatively
10.0
μ
g
negatively
negatively
5.0
μ
g
negatively
negatively
Further, to compare the anti-tuberculosis activity of
Biomayrin with isoniazid, ethambutol and rifampicin
anti-tuberculosis drugs, parallel to the solid nutrient
medium (Levenstein-Jensen), diagnostic materials
obtained from patients with a drug-sensitive form of
tuberculosis were inoculated into a modified MGIT
medium (Middlebrook 7H9) by adding BioMayrin.
Similarly as in solid nutrient medium, rifampicin,
isoniazid and ethambutol were added to individual
MGIT tubes. The prepared inoculums were incubated
in an automated test system BACTEC MGIT 960.
Interpretation of the mycobacterial colonies growth
was carried out using the built-in scanning algorithm
inside the incubator for 12 days.
The results of determination of drug susceptibility to
Biomayrin, rifampicin, ethambutol and isoniazid are
presented in table 4.
Table 4
The sensitivity of the MBT to Biomairin rifampicin, ethambutol and isoniazid
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Tested material
H37Rv (classical
strain)
16294 (patient)
16947 (patient)
The final concentration
of drugs
40,0
μ
g
Biomayrin
S
S
S
Rif
S
S
S
Etm
S
S
S
H
S
S
S
S- (sensitive) sensitive form
R- (resistant) stable form
As can be seen from table 4, all the studied drugs were
assessed as sensitive.
The sensitivity of drug-resistant strains obtained from
patients to the studied drugs was also investigated.
The concentration of drugs on liquid nutrient media
MGIT Middlebrook 7H9 was carried out in accordance
with the solid nutrient medium Levenstein-Jensen.
Dilutions of mycobacterial suspensions were prepared
in ratios according to McFarlan
d № 0.
5.
In order to obtain reliable results, diagnostic materials
obtained from patients with drug-resistant forms were
sown on solid nutrient medium (Levenstein-Jensen),
growth of M. tuberculosis in the media was observed
for 21 days. The results of tests for drug susceptibility
of resistant strains to the drug Biomayrin are shown in
Table 5.
Table 5
The sensitivity of the MBT to Biomayrin rifampicin, ethambutol and isoniazid
Biomayrin
The final
concentration of
drugs
11748
(patient)
11891
(patient)
К
1
-10
3
К
2
-10
4
К
1
-10
3
К
2
-10
4
40,0
μ
g
10 CFU
negatively
3 CFU
negatively
20,0
μ
g
8 CFU
1 CFU
2+
2+
Volume 02 Issue 12-2022
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10,0
μ
g
20 CFU
2+
3+
3+
5,0
μ
g
20 CFU
2+
3+
3+
Rif
40,0
μ
g
R
R
R
R
Etm
40,0
μ
g
R
R
R
R
H
40,0
μ
g
R
R
R
R
S- (sensitive) sensitive form
R- (resistant) stable form
As can be seen from table 5, resistant MBT strains
showed sensitivity only to Biomayrin among the
studied drugs at a concentration of 40
μ
g at a dilution
of
К
2
-10
4
, and in other cases showed resistance.
The comparative bacteriostatic activity of Biomayrin
with isoniazid and Mayrin anti-TB drugs (isoniazid - 75
mg, ethambutol - 300 mg; rifampicin - 150 mg) was
studied in vivo. The study was carried out on guinea
pigs and rabbits of the
“
Chinchill
a”
breed with virulent
Mycobacterium tuberculosis strains
“
Humanis NQ
2520
”
. The preparations were administered orally,
after 21 days of infection of animals with the
Mycobacterium tuberculosis strain "Humanis NQ
2520". Isoniazid and Mayrin in a dose of 10 mg/kg, and
Biomayrin based on the content of active substances.
The activity of the drugs was assessed by the index of
damage to the internal organs, which was determined
by the method of R. Vojtek (damage to the spleen):
IS =
М
S · 100%/
М
B,
where: IS - spleen index
MS - the mass of the spleen
MB
–
the mass of the div.
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The results of the study are presented in Figure 1.
As can be seen from Figure 1, Biomayrin and Mayrin
show pronounced anti-tuberculosis activity. In
particular, the lesion index of the spleen in control
animals is equal to 5.4%, with the introduction of
Biomayrin 0.32% and Mayrin equal to 0.24%.
Pathologicoanatomic studies of experimental animals
after the study showed that there were no significant
changes in the internal organs of animals taking
Biomayrin. And in animals taking Isoniazid and Mayrin,
there were some changes in the liver.
Toxicological studies of Biomayrin were compared
with low molecular weight analogues of isoniazid,
ethambutol and rifampicin. The results of the
experiments were processed by the method of
variation statistics. Calculations LD
50
were carried out
by the method of Litchfield and Wilcoxon. On the basis
of the conducted studies, the LD
50
of the studied drugs
were determined, which are presented in Table 6.
Table 6
Composition and LD
50
of the studied anti-TB drugs
Composition
Content of
isoniazid
Content of
ethambutol
Content of
rifampicin
LD
50
mg/kg
%
mole %
%
mole %
%
mole %
where: 1. Biomayrin; 2. Mayrin; 3. Control
Fig.1. Spleen lesion index.
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Isoniazid
100
100
-
-
-
-
224 (160÷300)
Ethambutol
-
-
100
100
-
-
1290 (1100÷1500)
Rifampicin
-
-
-
-
100
100
1570 (1350÷1800)
Biomayrin
15±3
13±3
15±3
10±3
17±3
5±2
≥
5000
As can be seen from table 6, the acute toxicity of
Biomayrin is much lower than that of low-molecular
analogs; in particular, LD
50
for Biomayrin was more
than 5000 mg/kg, whereas for isoniazid it was 224
mg/kg, ethambutol 1290 mg/kg and rifampicin 1570
mg/kg.
The pharmacokinetics of Biomayrin was studied in
comparison with Mayrin. Studies have shown that
after a single oral administration of Mayrin at a dose of
7.5 mg/kg, the drug is quickly absorbed, reaching a
maximum concentration (5.18 ± 0.54) in the blood after
1 hour, and in the lungs after 3 hours, and remains in
bacteriostatic range within 12 hours (Fig. 2 a-1). The
drug penetrates into all physiological fluids -
cerebrospinal fluid, pleural, ascitic, milk in nursing
mothers, and the level of its concentration in these
fluids is similar to its level in plasma.
With the introduction of Biomayrin in a dose of 15
mg/kg, isoniazid appears in the blood of rats after 60
minutes at a concentration (4.68 ± 0.57) and then the
concentration of isoniazid remains in the range of 3.0-
6.0 mcg/ml for 48 hours Then there is a gradual
decrease in the concentration of the drug in the blood
up to 72 hours (Fig. 2 a-2).
а
б
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с
Fig. 2.
Dynamics of changes in the concentration of a) isoniazid; b) ethambutol; c) rifampicin in serum after oral
administration of Mayrin (1) and Biomayrin (2).
The maximum concentration of ethambutol in serum
(6 µg/ml) is reached 2 hours after the administration of
Mayrin and after 24 hours its concentration is less than
1 µg/ml (Fig. 2. b-2).
With the introduction of Biomayrin, the maximum
concentration of ethambutol (5.83 µg/ml) in the blood
is observed 3 hours after administration. In the future,
the level of ethambutol in the blood gradually
decreases to 72 hours (Fig.2 b-2).
The maximum concentration of rifampicin in the blood
plasma after the administration of Mayrin is reached in
2-2.5 hours (6.3
μ
g/ml), it is found in therapeutic
concentrations in the pleural exudate, sputum,
contents of cavities, bone tissue, the greatest
concentration is created in the liver and kidneys. At the
therapeutic level, the concentration of the drug is
maintained for 8-12 hours (Fig. 2 b-1).
Based on the data obtained on the elimination of drugs
with urine from the div of rats, calculations of
pharmacokinetic parameters for Biomayrin in the
Borgia program were carried out. The results are
presented in table 7.
Table 7
Pharmacokinetic parameters of Biomayrin
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Pharmacokinetic parameters
Isoniazid
Ethambu
tol
R-cin
Biomayrin
Isoniazid
Ethambu
tol
R-cin
The period of semi-absorption
T1/2 (Ka), min
108.281
468.243
92.4
175.221
959.834
58.7
Half-life T1/(Kel), min
693.000
1420.08
752.2
742.765
2887.50
1626.7
Absorption constant Ka
0.00640
0.00148
0.007
0,00396
0.00072
0.01
Elimination rate constant Kel
0.00100
0.00049
0.0009
0.00093
0.00024
0.0004
The half-period of equilibrium
concentration T1/2 Css max,
min
0.290
0.366
0.25
0.341
0.366
0.105
Equilibrium concentration
Css max,
μ
g/ml
0.426
0.001
0.013
0.117
0.000
0.001
Volume of distribution Vd, ml
2173.90
47559.3
145.3
5763.53
305574
2108.7
Total clearance Clt ml/min
2.174
23.209
0.13
5.377
73.338
0.8
Area under
“
concentration-
time
”
curve AUG
230001
468.243
7465.2
92982,2
6817.8
1113.2
As seen with the introduction of Biomyirin, the
elimination half-life increases for all three isoniazid,
ethambutol, and rifampicin preparations. The rate of
elimination decreases. The period of semi-absorption
increases, the rate of absorption decreases. For
ethambutol, the equilibrium maximum concentration
does not change, for isoniazid and its modified forms,
the equilibrium maximum concentration decreases by
75%, and for rifampicin and its modified form, the
equilibrium maximum concentration decreases by 92%.
The total clearance of Biomayrin for isoniazid is
increased by 250% and for ethambutol it is increased by
320% and for rifampicin by 615%. This difference in
clearance is associated with greater bioavailability of
drugs in the composition of Biomayrin.
CONCLUSION
Thus, the results of the study showed that Biomayrin,
being less toxic than low-molecular analogs, has a
pronounced anti-tuberculosis effect. With a two-fold
lower dose in the calculation of the active substance in
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comparison with Mayrin, it exhibits almost identical
activity against the strain
М
B
Т
Humanis.
The results of pharmacokinetic studies have shown
that with the introduction of Biomayrin, the
therapeutic concentration of active substances
(isoniazid, ethambutol and rifampicin) in the blood
lasts longer than that of their low-molecular
counterparts. It should also be noted that the
introduction of isoniazid into the polysaccharide
matrix slows down its metabolism to the
therapeutically inactive acetylisoniazid. The results
obtained reliably confirmed the prolonged action of
Biomayrin.
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