ANTIPROFILERATIVE EFFECT OF EPURUBICIN - CHITOSAN CONJUGATES

N. Todorova, M. Ilarionova, D. T
Journal of the University of Chemical T
odorov
echnology and Metallurgy, 42, 3, 2007, 269-272
ANTIPROFILERATIVE EFFECT OF EPURUBICIN-CHITOSAN CONJUGATES
N. Todorova1, M. Ilarionova2, D. Todorov2
1University of Chemical Technology and Metallurgy
Received 21 March 2007
8 Kl. Ohridski, 1756 Sofia, Bulgaria
Accepted 12 July 2007
2National Centre of Oncology, Sofia 1756,
6 Plovdivsko pole blvd., Bulgaria
E-mail: todorova_nelly@abv.bg
ABSTRACT
The preparation and properties of conjugates of the antitumor antibiotic epirubicin/chitosan were investigated. An
in vitro study was performed on the =ntiproliferative activity of epirubicin and its conjugates EX1 and EX2. Sensitive HL-
60/S and medicine-resistant HL-60/DOX leucotic cell lines of mieloblastic origin were used. Comparison of the antitumor
effects of the two conjugates, EX1 and EX2, shows almost equal activities towards cell lines HL-60/S, which are much below
the activity of free epirubicin. With respect to the medicine-sensitive lineI HL-60/DOX the effect of the conjugates is more
pronounced than that of the free antibiotic. The better antiproliferative effect belongs to EX2.
Keywords: anthracycline antibiotics, epirubicin, epirubicin- chitosan conjugate, HL-60/DOX, HL-60/S cell lines,
MTT test.
INTRODUCTION
new remedies. It permits not only development of new
medicines but also an improvement of those already ex-
isting with a view to enhancing their activity and reduc-
The development of new methods and prepara-
ing the undesired secondary effects due to them [14,15].
tions in modern oncopharmacological practice is one
In this sense the conjugates of biologically active com-
of the fields in which present-day medicine has huge
pounds with such polymer carrier substrates represent a
achievements. Antibiotics with antitumor effect such as
new generation of products differing in nature and mecha-
the anthracyclines play an important role as remedies
nism of action from the initial substances [16,17].
against malignant new formations [1-3]. Having a high
In previous studies we investigated the biologi-
efficiency with respect to a series of tumors, these anti-
cal behaviour of the conjugates of a series of anthracyclic
biotics at the same time possess a significant
antibiotics bonded covalently to dextran or chitosan by
cardiotoxicity, which makes them inapplicable in large
various binding groups [11-13]. In all cases we observed
doses and for a longer period of time [4]. A series of
some properties favouring their application, e.g .a tox-
analogues of the anthracyclic antibiotics based on modi-
icity drop and an antitumor activity in doses much higher
fications of both the agluconic and the hydrocarbon part
than the nontoxic dose of free epirubicin. The use of
of the molecule have been prepared and investigated.
chitosan as a support is appropriate due to the presence
Many of them have found clinical application [5,8].
in its molecule of a considerable number of free hy-
Combining biologically active substances with
droxyl and amino groups which may be modified. In
biopolymers is one of the promising ways of obtaining
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Journal of the University of Chemical Technology and Metallurgy, 42, 3, 2007
addition, it is a biocompatible and biodestructable
Chitosan is a suitable carrier because it is
polymer. Some authors have also described a certain
biocompatible and biodestructable, and the
pharmacological effect of chitosan.
anthracyclinic tablets belong to the most active antitu-
mor agents.
The present investigation should establish
EXPERIMENTAL
whether the way of binding the active substance to the
The MTT test is described by Mosman in 1983.
matrix is of importance for its antitumor effect. In view
It is based on the reduction of the yellow tetrazole salt
of this we accomplished binding between the
MTT (3-)-4,5-dimethylthiasol-2-yl)-2,5-dimethyl-
anthracyclinic antibiotic epirubicin and chitosan by two
tetranol bromide to a violet formasan under the effect
different methods. According to the first method
of the mitohondrial dehydrogenases of the vital cells.
epirubicin was bonded to the matrix by reductive alky-
The MTT test was performed using a cell sus-
lation [5,12], i.e. binding was achieved by formation of
pension with a density of 3,5.10
a heterocyclic 7-atomic ring which contained an N atom
5 cells which was intro-
duced dropwise into 96 perforated plates (100 ml/hole).
from the amino group of the hydrocarbon residue of
After a 24 h incubation the cells were treated with solu-
the antibiotic (Fig. 1). The conjugate formed in this way
tions of the conjugates EX
had a stable bond between the matrix and the epirubicin.
1 and EX2 and epirubicin E,
10 ml MTT solution in a sterile phosphate buffer being
According to the other way of binding the antibiotic to
introduced to each hole after stirring the plates were
the matrix we realized formation of an azomethyn group
incubated at 37
between the keto group of the oxidized chitosan and
oC for about 4 h. During this time, the
vital tumor cells with functional mitohondries trans-
the amino group of the antibiotic. In this case epirubicin
formed the tetrazole component of MTT into a reagent
can be liberated from the conjugate molecule more eas-
with a pale yellow colour, which was partly insoluble in
ily [13] (Fig. 2).
the medium of cell cultures. To dissolve the formazan
Comparison of the results on the biological ef-
cells, 110 ml 5 % formic acid in isopropanol were in-
fect of the two conjugates can be an indication of the
troduced to each hole. The mixture was stirred intensely.
influence of the way of bonding on the biological effi-
Calorimetric purification was carried out at 580 nm
ciency of the conjugates.
using an Unicam I ELISA reader as well as at 620 nm,
An in vitro study was performed on the
as a periphery wave length. The absorption of the sub-
antiproliferative activity of epirubicin and its conjugates
stances in the holes containing treated tumor cells was
EX1 and EX2. Sensitive HL-60/S and medicine-resis-
compared with that of untreated controls. During the
tant HL-60/DOX leucotic cell lines of mieloblastic ori-
calculation of the experimental results the percentage
gin were used.
of the vital cells was found using the formula
The preparations were applied over a wide range
% vital cells = (A /A ).100
of concentrations: (2 mg/ml - 1250 mg/ml) for incuba-
t
c
where A is the sample absorption and A , the absorp-
tion times of 48 and 72 h. A distinct relationship be-
t
c
tion of untreated control
tween concentration and cell structure suppression was
observed. The average amount of preparations (IC ),
50
ensuring 50 % cell growth suppression with the cell
RESULTS AND DISCUSSION
lines under consideration and confidence values IC
((a=0,05, S2n=1.) found by a semi-automatic MTT test
The preparation and properties of conjugates of
was determined. The results of the investigations are
the antitumor antibiotic epirubicin/chitosan were in-
given in Table 1. It is evident that the values obtained
vestigated. Introduction of biologically active com-
for the conjugate EX1 are 21,60 mg/ml HL-60/S and
pounds to carriers is usually accompanied by a decrease
20.43 mg for the resistant cells, while epirubicin has a
in toxic effect and the appearance of new useful proper-
pronounced effect on the sensitive cells, HL-60/S. The
ties such as: directed transport of the remedy to the
results obtained show that the conjugate of epirubicin
target of its action as well as prolongation of the action.
is less active than the free epirubicin. This may be due
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N. Todorova, M. Ilarionova, D. Todorov
Table 1. Antiprofilerative effect of Epurubicin-Chitosan
shows that the effect of the conjugate differs in charac-
Conjugates.
ter from that of epirubicin. Similar results have been
obtained with in vitro experiments on lymphoid leuco-
sis L1210 and lymphocytic leucosis P388 performed
Cell lines
Substance
Incubation
IC50
IC50
times
[ g/ml]
[µM/ml]
on animals. The dose was introduced on the 1th, 4th and
HL-60 S
Epirubicin
72
0,41
0,71(0,54-
0,84)
8th day after the tumor transplantation. The results of
HL-60 S
EX2
48
17,34
23,7(22,6-
the experiments are shown in Table 2. It is obvious that
72

24,7)
32,62
44,5(42,2-
free epirubicin shows its strongest effect in doses of 0.25
46,7)
HL-60 DOX
Epirubicin
48
6,73
11,6(11,5-
mg/kg. With doses up to 1 mg/kg this effect decreases
11,7)
HL-60 DOX
EX2
48
18,24
24,3(23,6-
and with 2 mg/kg a toxic effect is observed. Experi-
72

24,9)
13,46
22,6(22,5-
ments with free chitosan on lymphocytic leucosis P388
22,7)





have shown no antitumor effect of chitosan even with
HL-60 S
EX1
48
21,60
29,5(25,8-
doses of 500 mg/kg. Simultaneously, the EX1 and EX2
72

33,2)
37,54
51,6(50,5-
conjugates have displayed an antitumor effect with doses
52,7)





of 1 to 18 mg.kg. Both models of tumor lines show the
HL-60 DOX
EX1
48
20,43
27,9(25,5-
72

30,5)
highest antitumor effect with 18 mg/kg doses.
13,46
19,9(19,5-
20,3)
The value of the T/C criterion for EX1 with a

dose of 18 mg/kg is 27.1 % for L1210 and 281.4 % for
Table 2. Antitumor effect of epirubicin, chitosan and
P388. The presence of an antitumor effect of the conju-
conjugate of epirubicin with chitosan EX1 and EX2 on
gate in experiments on animals is most probably due to
leucosis L 1210 and leucosis P388.
the liberation of the antibiotic from the matrix and its
Substance
Dose [mg/kg]
Leukosis
Leukosis P 388
direct effect on the tumor cells according to the
L1210[T/C,%]
[T/C,%]

intercallation method characteristic of anthracyclines.
Epirubicin
0,25
176,1
176,1
Another positive result of the present investigation is
Epirubicin
0,50
159,4
159,4
Epirubicin
1,0
98,3
98,3
that an antitumor effect is observed over a wide range
Epirubicin
2,0
97,4
97,4
of concentrations, from 1 to 18 mg/kg, which is of great
Chitosan
125
98,5
98,5
Chitosan
250
88,6
87,4
importance for the therapeutical practice.
EX1
1,0
132,7
159,2
Table 2, presenting the results on the antitumor
EX1
2,0
166,4
217,1
EX1
4,0
281,4

activity of EX2, shows the highest T/C values for 18
EX1
18
274,1
102,1
mg/kg doses again, these values amounting to 372 % for
EX1
36,0
121,3
98,2
L1210 and 384.1 % for P 388.
EX2
1,0
143,8
151,1
EX2
2,0
176,7
178,8
Comparison of the antitumor effects of the two
EX2
4,0
298,3
237,4
conjugates, EX1 and EX2, shows almost equal activities
EX2
18,0
379,2
384,1
EX2
36,0
87,1
112,4
towards cell lines HL-60/S, which are much below the

activity of free epirubicin. With respect to the medi-
to the effect of the conjugates on the plasma membrane
cine-sensitive lines HL-60/DOX the effect of the conju-
and not to the biological effect of the anthracyclines
gates is more pronounced than that of the free antibi-
through intercallation. The conjugates obtained have a
otic. The better antiproliferative effect belongs to EX2.
more pronounced effect on resistant cell lines HL-60/
This may be due to the fact that in the case of EX1 the
DOX than is the case of sensitive lines HL-60/S. In the
liberation of epirubicin from the matrix leads to reap-
study on the antiproliferative effect of the conjugate EX2,
pearance of the initial antibiotic while with EX2 the
the following values of IC were obtained: 32.62 mg/
50
reductive alkylation results in a 7-atomic ring as a bond
ml for 72 h and 17.34 mg/ml for 48 h with HL-60/S ;
between matrix and antibiotic, and the conjugate affects
18.24 mg for 28 h and 13.4 mg/ml for 72 h with HL-
directly the membrane of the tumor cells.
60/DOX (6.73 for free epirubicin).
In experiments on test animals both conjugates
Obtaining a result that shows a less pronounced
show a maximum antitumor effect in amounts of 18
effect of the conjugates than that of the free antibiotic
mg/kg, in this case again the conjugate EX2 having a
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Journal of the University of Chemical Technology and Metallurgy, 42, 3, 2007
Fig. 1. Proposed chemical way for epirubicin conjugation to
Fig. 2. Proposed chemical way for epirubicin conjugation to
chitosan (EX2).
activated chitosan (EX1).
stronger antitumor effect than that of EX1. The values of the
4. C. Depred, D. De Campeneere, Eur. J. Cancer, 16, 8,
criterion T/C are 379.2 % for EX2 in the case of L1210
1980, 981-986.
(274/1 % for EX1) and 384.1 % for EX2 in the case of P388
5. R. Borch, M. Benstein, D. Dupont, J. Am. Chem.
(274.1 % for EX1). These results show that the binding of
Soc., 93, 12, 1971, 2897-2902.
epirubicin by reductive alkylation is preferable to the other
6. T. Coviello, M. Dentini, G. Rambone, P. Desideri,
case where binding is achieved by an azomethyn group.
M. Carafata, E. Murtas, F.M. Riccieri, F.A.
Alhaique, J. Controlled Release, 55, 1, 1971, 57-
CONCLUSIONS
66.
7. M. Inaba, R.K. Johnson, Biochem. Pharmacol., 27,
• The antiproliferative effects of two conjugates of
1998, 2123-2130.
epirubicin with chitosan and of free epirubicin with
8. F. Fresard, A. Garnier-Suillerot, Biochim. Biophys.
respect to cell lines HL-60/S and HL-60/DOX (both
Acta, 10, 36, 1990, 121-127.
of mieloidic origin) have been investigated.
9. M. Frezard, A. Garnier-Suillerot, Biochim. Biophys.
• A direct antiproliferative effect has been established
Acta, 10, 1991, 29-35.
with all compounds by determining the mean inhib-
10. M. Fiallo, A. Laigle, M. Borrel, A. Garnier, Bio-
iting concentration (IC ) for 48 and 72 h incubation.
chemical Pharmacology, 45, 3, 1993, 659-665.
50
11. N. Todorova, M. Maneva, M. Ilarionova, A. Dudov,
• A high antitumor activity has been shown by both
D. Todorov, Biotechnol Biotechnol Eq., 2, 17, 2003,
conjugates (EX1 and EX2) with respect to lymphoid
151-153.
leucosis L1210 and lymphocytic leucoses P 388. The
12. N. Todorova, M. Krysteva, K. Maneva, D. Todorov,
highest value of the T/C criterion is observed with
J. Bioactiv Comp. Polym., 1999,178-184.
samples of 18 mg/kg.
13. N. Todorova, M.Ilarionova, K.Todorov, D.Todorov;
• The conjugate EX2 has shown a more pronounced an-
Biotechnol Biotechnol Eq., 2, 18, 2004, 123-129
titumor activity than that of EX1.
14. F. Alhaique, E. Santucci, M. Carafata, T. Coviello,E.
Murtas, F.M. Riccieri, Biomaterials, 17, 20, 1996,
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