Polymer conjugates of the highly potent cytostatic drug 2-pyrrolinodoxorubicin

Article abstract of DOI:10.1016/j.ejps.2010.11.006

This paper describes the synthesis and biological evaluation of a conjugate of the highly cytotoxic drug 2-pyrrolinodoxorubicin (p-DOX) with an N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) as a water-soluble biocompatible polymer carrier, utilizing the advantageous concept of polymer-drug conjugates. The conjugate of p-DOX with HPMA copolymer (PHPMA/p-DOX) was prepared by reacting the PHPMA/DOX conjugate, where the DOX was bound via a hydrazone bond, with 4-iodobutyraldehyde. The hydrazone bond between the polymer and drug is susceptible to pH-controlled hydrolysis, enabling prolonged stability in circulation and fast p-DOX release under conditions mimicking the intracellular environment. The in vitro cytostatic activity of free p-DOX was in accordance with literature, whereas its PHPMA conjugate exhibited a 1.3- to 5-fold lower cytotoxicity, depending on the cancer cell line, when compared to the free p-DOX. This is in qualitative agreement with the data obtained for DOX and its HPMA copolymer conjugates. On mice bearing T-cell EL4 lymphoma, no tumor suppression was observed from the free p-DOX at a subtoxic dose of 0.1 mg/kg, whereas the PHPMA/p-DOX conjugate significantly inhibited the initial tumor growth at approximately equitoxic doses of 0.4 and 0.8 mg p-DOX eq/kg. However, moderately elevated doses of the p-DOX equivalent in the conjugate caused toxic effects, making accurate dosage setting essential.

Full text of DOI:10.1016/j.ejps.2010.11.006

European Journal of Pharmaceutical Sciences 42 (2011) 156–163
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Polymer conjugates of the highly potent cytostatic drug 2-pyrrolinodoxorubicin
a,∗
a
b
b
M. Studenovsky , K. Ulbrich , M. Ibrahimova , B. Rihova
a
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic
b
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the synthesis and biological evaluation of a conjugate of the highly cytotoxic drug
Received 16 September 2010
Received in revised form 3 November 2010
Accepted 8 November 2010
Available online 12 November 2010
2
-pyrrolinodoxorubicin (p-DOX) with an N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) as a
water-soluble biocompatible polymer carrier, utilizing the advantageous concept of polymer–drug con-
jugates. The conjugate of p-DOX with HPMA copolymer (PHPMA/p-DOX) was prepared by reacting the
PHPMA/DOX conjugate, where the DOX was bound via a hydrazone bond, with 4-iodobutyraldehyde.
The hydrazone bond between the polymer and drug is susceptible to pH-controlled hydrolysis, enabling
prolonged stability in circulation and fast p-DOX release under conditions mimicking the intracellular
environment. The in vitro cytostatic activity of free p-DOX was in accordance with literature, whereas its
PHPMA conjugate exhibited a 1.3- to 5-fold lower cytotoxicity, depending on the cancer cell line, when
compared to the free p-DOX. This is in qualitative agreement with the data obtained for DOX and its
HPMA copolymer conjugates. On mice bearing T-cell EL4 lymphoma, no tumor suppression was observed
from the free p-DOX at a subtoxic dose of 0.1 mg/kg, whereas the PHPMA/p-DOX conjugate significantly
inhibited the initial tumor growth at approximately equitoxic doses of 0.4 and 0.8 mg p-DOX eq/kg. How-
ever, moderately elevated doses of the p-DOX equivalent in the conjugate caused toxic effects, making
accurate dosage setting essential.
Keywords:
Polymer conjugates
Tumor drug delivery
pH-controlled release
Anticancer agents
Multidrug resistance
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
their biological activity to date. Despite their partial selectivity to
rapidly dividing cells (tumors), in most cases their non-specific
toxicity limits their clinical use. In order to reduce some side
effects of cytotoxic chemotherapeutics, many strategies have been
developed, including a combination of different drugs in one trial,
specific time schedule, treatment adapted to the exact diagno-
sis and modification of drug formulation. In addition, it has been
observed that when the parent drug is modified with a natural
or synthetic water-soluble polymer, a significant suppression of
adverse effects is achieved. Moreover, the solid tumor specificity
of such polymer–drug conjugates is dramatically increased thanks
to the EPR effect (Maeda et al., 2000; Matsumura and Maeda, 1986;
Seymour et al., 1995).
Polymer conjugates of anthracyclines have been known for
approximately 25 years (Duncan et al., 1987; Kopecek and Duncan,
1987; Rihova and Kopecek, 1985). PHPMA/DOX conjugates with
enzymatically degradable spacers were one of the first conjugates
synthesized (Duncan, 2009). These conjugates exhibited greatly
reduced toxicity, but relatively high doses were required for an
effective treatment. We have overcome this drawback by replacing
doxorubicin with its more potent anthracycline derivative.
A new class of anthracycline analogs has emerged as a result of
the strong effort to synthesize more efficient derivatives of doxoru-
bicin (Binaschi et al., 2001). The formation of reactive immonium
ions in slightly acidic media is the key feature of these compounds.
The clinical treatment of cancer relies on the complementary
effects of surgery, immunotherapy, radiotherapy and chemother-
apy. The latter approach involves treatments using a broad variety
of synthetic or natural substances that exhibit specific tumor-
suppressive properties. The anticancer activity of such compounds
is derived from a number of particular effects resulting in the
death of tumor cells; their most common mode of action is a
direct cytotoxic effect based on the interruption of the tumor cell
cycle, inducing cell apoptosis. Anthracyclines are typical repre-
sentatives of such cytotoxic agents. The anti-prolific activity of
anthracyclines arises mainly from their strong interaction with
nuclear DNA, disabling its transcription during the cell cycle. Dox-
orubicin is the most frequently used drug in this family. Despite its
high potency and moderate side effects, a strong effort has been
made towards the additional improvement of its therapeutic pro-
file. There are several approaches to this optimization, including
the modification of drug formulation and the chemical deriva-
tion of the parent compound. In response, hundreds of chemical
analogs of doxorubicin have been synthesized and evaluated for
∗ Corresponding author. Tel.: +420 296 809 230; fax: +420 296 809 410.
E-mail address: studenovsky@imc.cas.cz (M. Studenovsky).
0
928-0987/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2010.11.006

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
157
O
O
OH
O
O
OH
O
OH
OH
OH
OH
O
OH
O
O
OH
O
O
1.H+
. rearrangement
O
O
2
OH
OH
+
N
N
2
-pyrroline ring
immonium ion
Scheme 1. Formation of reactive immonium cation in p-DOX.
Because the immonium group is a strong electrophile, it has a
strong ability to bond covalently with DNA. As the main thera-
peutic effect of DOX is derived from its interaction with nuclear
DNA inside the tumor cell, its cytostatic activity is thus dramati-
cally increased. Moreover, these compounds exhibit much lower
cardiotoxicity and multidrug resistances (MDR), which are usu-
ally the most serious limitations in therapy with doxorubicin.
and multi-angle light-scattering DAWN DSP-F detectors (Wyatt,
USA).
UV/vis spectra were measured on a SPECORD 205 Spectrometer
(Analytik Jena AG, Germany).
2
2
.3. Syntheses
2
-Pyrrolinodoxorubicin, an anthracycline analog that is approx-
.3.1. Preparation of 2-(3-iodopropyl)-1,3-dioxolane
imately 100 times more potent than DOX in vitro, is one of the
members of this family (Scheme 1). The aim of this study was to
synthesize a PHPMA/p-DOX conjugate with pH-sensitive hydra-
zone linkage and then evaluate the conjugate’s biological activity
in vitro and in vivo.
2
-(3-Chloropropyl)-1,3-dioxolane (15.8 g, 0.1 mol) was dis-
solved in 150 ml of acetone that contained 30 g (0.2 mol) of NaI and
.5 g of NaHCO3 as a stabilizer. The solution was refluxed for 20 h
2
and then evaporated to dryness. The obtained solid was washed
three times with 100 ml of diethyl ether, filtered off and the yel-
lowish filtrate was stirred with 20 g of anhydrous Na SO , 1 g of
2
4
Na S O and 1 g of NaHCO3 for 12 h. The mixture was filtered off
2
. Materials and methods
2
2
3
and 100 mg of Na CO3 was added to the filtrate with following
2
removal of diethyl ether under vacuum. The crude oily product
was purified by distillation under reduced pressure and stored in
2
.1. Chemicals
◦
the dark at −18 C with a small amount of fine-grained Na CO
1
-Aminopropan-2-ol,
methacryloyl
chloride,
2,2-
2
3
as a stabilizer. The purity of the colorless liquid product was con-
azobisisobutyronitrile (AIBN), N,N-dimethylformamide (DMF),
N-ethyldiisopropylamine (DIPEA), dimethyl sulphoxide (DMSO),
trifluoroacetic acid (TFA), 6-aminohexanoic acid, methyl
firmed by HPLC; yield: 23.4 g (92%); elemental analysis: calcd. C,
1
2
9.77; H, 4.58; I, 52.43%; found C, 30.04; H, 4.62; I, 52.09%; H NMR
3
00 MHz (CDCl , 297 K): 1.78 m (2H, I–CH CH CH ), 1.97 quint.
6
-aminohexanoate hydrochloride, hydrazine hydrate, 2-(3-
3
2
2
2
(
2H, I–CH CH CH ), 3.24 t (2H, I–CH CH CH ), 3.89 dt (4H, O–CH ),
chloropropyl)-1,3-dioxolane, sodium iodide, sodium thiosulphate,
sodium acetate, DOX hydrochloride (DOX·HCl) and silica gel were
purchased from Aldrich. Acetonitrile and other common solvents
and chemicals were purchased from Lach-Ner, s.r.o.
2 2 2 2 2 2 2
4
.90 t (1H, O–CH–O).
2.3.2. Preparation of 4-iodobutyraldehyde
-(3-Iodopropyl)-1,3-dioxolane (6 g, 24.8 mmol) was dissolved
2
in 240 ml of tetrahydrofuran, and then a solution of 40 ml of concen-
trated hydrochloric acid in 800 ml of water was added. An initially
cloudy reaction mixture was stirred in the dark at r.t. for 24 h and
then extracted five times with 50 ml of dichloromethane. The com-
2
.2. Analytical methods
HPLC analyses were performed on a HPLC chromatograph (LDC
Analytical, USA) using a reverse-phase column (Chromolyth Per-
formance RP-18e 100 × 4.6 mm) with UV detection. A mixture of
water–acetonitrile was used as the eluent at a gradient 0–100 vol%
and a flow rate of 2 ml/min.
Elemental composition was determined using a Perkin Elmer
Elemental Analyzer 2400 CHN (Perkin Elmer, USA).
bined organic layers were dried with anhydrous Na SO4 and a
2
small amount of NaHCO , the solids were removed via filtration and
3
the solvent was evaporated under reduced pressure with an addi-
tional portion of NaHCO3 as a stabilizer. Finally, a small amount
of fine-grained Na S O was added as an iodine scavenger. The
2
2
3
◦
stabilized 4-iodobutyraldehyde was stored in the dark at −18 C.
Melting point temperatures were determined on a Kofler’s block
Purity of the colorless liquid product was confirmed by HPLC; yield:
(
VEB Analytik Dresden, Germany).
4
.4 g (90%); elemental analysis: calcd. C, 24.26; H, 3.56; I, 64.09%;
NMR spectra were measured on a Bruker Avance MSL 300 MHz
1
found C, 24.06; H, 3.40; I, 63.85%; H NMR 300 MHz (CDCl , 297 K):
NMR spectrometer (Bruker Daltonik, Germany).
MALDI-TOF spectroscopy was carried out using a Bruker Bifex
III Mass Spectrometer (Bruker Daltonik, Germany).
3
2
.13 quint. (2H, I–CH CH CH ), 2.63 t (2H, I–CH CH CH ), 3.23 t
2 2 2 2 2 2
(
2H, I–CH CH CH ), 9.80 s (1H, CH O).
2 2 2
Molecular weights of the polymers were determined by gel
permeation chromatography (GPC) in a mixture of acetate buffer
2.3.3. Preparation of p-DOX
(
pH 6.5, 0.3 mol/l) and methanol (20:80, v/v) on a TSK 3000 col-
To a solution of doxorubicin hydrochloride (100 mg, 0.17 mmol)
in 10 ml of ethanol was added 4-iodobutyraldehyde (270 mg,
1.4 mmol) and NaHCO3 (0.9 g, 10.7 mmol). The reaction mixture
umn (Polymer Laboratories Ltd., UK) using an HPLC System ÄKTA
Explorer (Amersham Biosciences, Sweden) equipped with RI, UV

1
58
M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
was diluted with 10 ml of dichloromethane and stirred in the
dark at r.t. for 4 h. The suspension was filtered off, the filtrate
was diluted with 50 ml of hexane and the precipitate that formed
was separated by centrifugation. The obtained solid was subse-
quently re-dissolved in 5 ml of methanol containing 100 ␮l of
trifluoroacetic acid and the solution was diluted with 100 ml of
ethyl acetate and concentrated under reduced pressure to approx-
imately one-tenth of the original volume. The obtained p-DOX
high-molecular-weight peak areas in GPC UV/VIS chromatogram
at 480 nm): <0.1 wt%.
2.3.8. Preparation of PHPMA/p-DOX conjugate
4-Iodobutyraldehyde (108 mg, 0.55 mmol) was added to a solu-
tion of PHPMA/DOX (103 mg, 11.4 ␮mol of DOX equivalent) and
anhydrous sodium acetate (190 mg, 2.3 mmol) in 4 ml of methanol,
and the reaction mixture was stirred for 2 h. The resulting solution
was diluted with 4 ml of methanol and subjected to separation by
chromatography on a Sephadex LH-20 column using methanol as
the eluent. The PHPMA/p-DOX conjugate was isolated as a high-
molecular-weight fraction, concentrated under reduced pressure
and precipitated into dried diethyl ether. The suspension was
centrifuged and the precipitated product was dried under vac-
uum. Yield: 80 mg (76%); molecular weight (GPC): Mw = 48 kDa,
Mw/Mn = 2.2; p-DOX/DOX conversion rate (HPLC analysis of com-
pletely hydrolyzed conjugate in 5% aqueous TFA): 95%; total p-DOX
content (calculated from the p-DOX/DOX conversion rate): 6 wt%;
free p-DOX content (calculated as a ratio between the correspond-
ing low-molecular-weight and high-molecular-weight peak areas
in GPC UV/VIS chromatogram at 480 nm): <0.1 wt %.
◦
trifluoroacetate was left to crystallize at −18 C overnight, sep-
arated by centrifugation and then dried under vacuum. Purity
of the dark-red crystalline product was confirmed by HPLC;
yield: 85 mg (78%); MALDI-TOF MS: calcd. 595, found 595.98
(
M+H).
2
(
.3.4. Preparation of N-(2-hydroxypropyl)methacrylamide
HPMA)
Synthesis of this monomer was carried out according to the lit-
erature procedure (Ulbrich et al., 2000). Yield: 32 g (75%); elemental
analysis: calcd. C, 58.72; H, 9.15; N, 9.78%; found C, 58.90; H, 9.10;
◦
N, 9.88%; m.p. 69–71 C.
2
(
.3.5. Preparation of 6-methacrylamidohexanohydrazide
MA-HH)
Synthesis of this reactive co-monomer was carried out using
a two-step literature procedure (Hruby et al., 2007). Yield: 5.6 g
2
2
.4. In vitro and in vivo studies
.4.1. Cancer cell lines
Two cancer cell lines of mouse origin (T cell lymphoma EL 4
(
80%); elemental analysis: calcd. C, 56.32; H, 8.98; N, 19.70%; found
and B cell lymphoma 38C13) and two cell lines of human origin
UKF-NB4 and UKF-NB4/Dox) were used in this study. Mice cancer
◦
C, 56.49; H, 8.63; N, 19.83%; m.p. 77–79 C.
(
cell lines were purchased from American Type Culture Collection
2
.3.6. Preparation of copolymer of
◦
(ATCC, Rockville, MD, USA) and grown in cultivation flasks at 37 C
N-(2-hydroxypropyl)-methacrylamide with
with 5% CO2 in RPMI 1640 medium (Gibco Laboratories) supple-
mented with heat-inactivated 10% fetal calf serum (FCS), 2 mM
l-glutamine, 50 mM 2-mercaptoethanol, 4.5 g/l glucose, 1.0 mM
sodium pyruvate, 100 U/ml penicillin and 100 ␮g/ml streptomycin.
Human cancer cell lines were the kind gift of Professor
Tomas Eckschlager (Pediatric Clinic, University Hospital Motol,
Prague, Czech Republic). They were established from human
surgical samples (neuroblastoma) and maintained as doxorubicin-
sensitive (UKF-NB4) or doxorubicin-resistant (UKF-NB4-DOXO)
clones (Bedrnicek et al., 2005). Cancer cell lines were grown
in IMDM medium (Gibco Laboratories) supplemented with 10%
fetal calf serum (FCS), 2 mM l-glutamine, 100 U/ml penicillin and
6
-methacrylamidohexanohydrazide (poly(HPMA-co-MA-HH))
HPMA (3.2 g, 22.4 mmol), MA-HH (165 mg, 0.77 mmol) and AIBN
(
150 mg, 0.91 mmol) were dissolved in 20 ml of methanol and dry
nitrogen was bubbled through the solution for 20 min. The solu-
tion was then heated in a sealed glass ampoule at 60 C for 17 h.
◦
The copolymer was precipitated into ethyl acetate, filtered off
and purified by recrystallization from methanol into ethyl acetate.
The suspension was filtered off and the resulting copolymer was
dried under vacuum. Yield: 2.7 g (80%); molecular weight (GPC):
Mw = 30 kDa, Mw/Mn = 2.2; hydrazide content (TNBSA assay (Etrych
et al., 2001)): 0.14 mmol/g; molar ratio of HPMA: MA-HH monomer
units (calculated from the hydrazide content) ∼98:2.
1
00 ␮g/ml streptomycin. The doxorubicin-resistant subline was
kept at 20 ␮g/ml of doxorubicin.
2.3.7. Attachment of DOX to poly(HPMA-co-MA-HH)
DOX·HCl (98 mg, 0.17 mmol) was added to a solution of
2
.4.2. Experimental animals
Inbred strains of C57BL/6 (H-2 ) mice, aged 8–12 weeks, were
poly(HPMA-co-MA-HH) (470 mg, 0.066 mmol of hydrazide groups
equivalent) and 200 ␮l of acetic acid in 3 ml of methanol and
the reaction mixture was stirred in the dark at r.t. for 48 h. The
unreacted DOX was then removed by centrifugation and the result-
ing solution was diluted with 5 ml of methanol and subjected
to separation by chromatography on a Sephadex LH-20 column
using methanol as the eluent. The PHPMA/DOX conjugate was
isolated as a high-molecular-weight fraction, concentrated under
reduced pressure and precipitated into dried diethyl ether. The
suspension was centrifuged and the precipitated dark-red pow-
der was dried under vacuum. Yield: 450 mg (88%); molecular
weight (GPC): Mw = 40 kDa, Mw/Mn = 2.2; total DOX content (deter-
mined spectrophotometrically in methanol solution at 480 nm;
b
purchased from the Animal Centre of the Institute of Physiology,
Academy of Sciences of the Czech Republic, v.v.i. All mice were
housed in accordance with approved guidelines and were provided
with food and water ad libitum. All studies were approved by the
Institutional Review Board.
2.4.3. In vitro cytostatic activity
The cytostatic potential of the conjugates was assessed using
3
a [ H]-thymidine incorporation assay. NUNCLON 96-well, flat-
3
bottomed plates were seeded with 5 × 10 /well of EL4 cells,
3
4
2
1
× 10 /well of 38C13 cells, 1 × 10 /well of UKF-NB4 cells and
4
× 10 /well of UKF-NB4-DOXO cells. The samples were tested in
1
−1
−1
ε = 11200 l mol cm ): 6.2 wt%; free DOX content (calculated
as a ratio between the corresponding low-molecular-weight and
triplicate or quadruplicate and then added to the wells to achieve
the desired concentrations. The plates were cultured in 5% CO2 for
7
◦
3
2 h at 37 C. For the last 6 h of the incubation, 18.5 kBq of [ H]-
thymidine was added per well. The cells were then collected onto
glass fiber filters (Filtermat, Wallac, Finland) using a cell harvester
(Tomtec, Orange, CT). After drying, the fiber filter was placed into
1
ε value was determined previously for conjugated DOX (Pillai and Panchagnula,
001).
2

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
CH3 CH3
159
CH2
CH2
O
O
a
b
NH
NH
CH2
(CH2)5
CH OH
CH3
O
NH
N
O
O
OH
OH
OH
OH
O
O
O
OH
NH2
H O, pH 7.4 (slowly)
H O, pH 5.5 (quickly)
2
2
O
CH3
CH3
CH2
O
O
OH
CH2
OH
O
O
b
OH
a
+
NH
NH
CH2
(CH2)5
O
OH
O
CH OH
CH3
O
O
NH
NH2
OH
NH
2
Scheme 2. Release of DOX from the polymer conjugate. At pH 7.4, only 10% of free DOX was released vs. 90% at pH 5.5 after 24 h. a:b ∼ 98:2.
a sample bag, a solid scintillator-Meltilex (Wallac) was applied
and the bags were sealed (Microsealer, Wallac). Counting was per-
formed in a 1450 MicroBeta TriLux (Wallac). Cells cultivated in
fresh medium were used as controls.
3. Results and discussion
In this report, we show the possibility of utilizing a water-
soluble synthetic polymer as a carrier for 2-pyrrolinodoxorubicin
(p-DOX), a highly potent cytotoxic analog of doxorubicin. The ben-
efits of the concept of polymer–drug conjugates have been proven
extensively, encompassing a wide range of therapeutics conjugated
with synthetic polymers (Pillai and Panchagnula, 2001). In our
group, HPMA copolymer-based conjugates have been investigated
in detail, and doxorubicin has been particularly studied as a drug.
The hydrazone-type PHPMA/DOX conjugate strategy provides the
usual profits of polymer conjugates as well as the advantageous
drug pharmacokinetics: the hydrazone linkage between DOX and
the polymer is sufficiently stable at pH ∼ 7.4 (blood stream) but
quickly hydrolyzed at pH ∼ 5–6 (cell endosomes and secondary
lysosomes) (Scheme 2). Consequently, the circulation period of the
conjugated DOX in the body is dramatically increased compared
with its free form, but the free drug is rapidly released inside the
tumor. Therefore, this scheme was applied as a design for a polymer
p-DOX conjugate.2
2
.4.4. In vivo tumor model
C57BL/6 (B/6; H-2 ) females were subcutaneously transplanted
b
5
once with a lethal dose (1 × 10 ) of EL4 T cell lymphoma cells,
a lethal dose being defined as that which induces tumor growth
in all controls. The mice that developed palpable tumors reach-
ing 5–8 mm in diameter within 8–9 days after the implantation
were intravenously treated with polymer conjugates diluted in
PBS, as described in Section 3. Those surviving at least 60 days
without any signs of tumor were considered long-term survivors
(
LTS), re-transplanted with a lethal dose of the same tumor cells
and left without treatment to determine therapy-induced tumor
resistance.
2
.4.5. Acute toxicity
A symptom of acute toxicity was determined as a decrease of
body weight in the experimental animals in the days following
treatment.
2
The <fn0010>kinetics of hydrolysis of the PHPMA conjugate could not be easily
determined in in vitro model system because of freed p-DOX re-attaching to the
polymer via coupling of p-DOX immonium groups (Scheme 1) with the polymer
hydrazide groups (data not shown). As stated below, chemical modification of the
daunosamine amino group in DOX molecule can only negligibly affect reactivity of
the outlying hydrazone bond. Thus, we expect the same release profile for DOX and
p-DOX from the polymer carrier, i.e. 10% release at pH 7.4 and 90% release at pH 5.5
within 24 h.
2
.4.6. Statistical analysis
All in vivo experiments were repeated at least once, and similar
results were obtained. The Student ˇı s t-test was used for evaluating
statistical significance. P < 0.05 was considered to be a significant
value.

1
60
M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
2
1
-(3-chloropropyl)-
,3-dioxolane
2-(3-iodoropropyl)-
1,3-dioxolane
4-iodobutyraldehyde
O
O
+
KI
acetone
H O , H
2
Cl
I
O
I
O
O
Scheme 3. Synthesis of 4-iodobutyraldehyde.
3.1. Synthesis
HPMA
MA-HH
CH3
An improved synthesis of 4-iodobutyraldehyde, the key sub-
CH3
stance needed for the synthesis of p-DOX, is described. Although a
two-step preparation procedure of this synthesis was published
CH2
CH2
O
O
(
Nagy et al., 1996), its difficult reproducibility and the massive
NH
NH
formation of various side-products using this procedure were
major obstacles. These complications were observed predomi-
nantly during the isolation of 4-iodobutyraldehyde in the second
synthetic step, but also to a lesser extent after the conversion of 2-
+
CH2
(CH2)5
CH OH
O
NHNH2
CH3
(
(
3-chloropropyl)-1,3-dioxolane to 2-(3-iodopropyl)-1,3-dioxolane
Scheme 3). In an attempt to synthesize the 4-iodobutyraldehyde
in sufficient yield and purity, the synthesis was conducted repeat-
edly under variable, well-controlled conditions. While doing these
experiments, we observed a self-acidification of freshly prepared
polymerization AIBN, 60°C
CH3
CH3
4
-iodobutyraldehyde, where the pH value of its aqueous emul-
sion was ∼4.5. Since aldehydes are potential substrates for an
acid-catalyzed aldol reaction (Smith and March, 2001), we consid-
ered this as a possible dominant source of side-reactions. Traces
of hydrogen iodide, formed by an elimination reaction from 4-
iodobutyraldehyde, could be a source of acid needed for the
initialization of aldol condensation. Therefore, a small amount of
NaHCO3 was added to a reaction mixture in order to neutralize
this acid. This modification led to a proper reaction course with
excellent yields in both reaction steps and was, in turn, proposed
as a final synthesis of 4-iodobutyraldehyde.
CH2
CH2
O
O
NH
a
NH
b
CH2
(CH )
2 5
CH OH
O
NHNH2
CH3
poly(HPMA-co-MA-HH)
p-DOX, in its free form as a trifluoroacetate, was prepared by the
modified synthesis described in literature (Nagy et al., 1996). The
original procedure involved the use of N-ethyldiisopropylamine
Scheme 4. Synthesis of the polymer precursor by the copolymerization of
monomers. a:b ∼ 98:2.
(
4
DIPEA) as a base during the alkylation step of DOX with
-iodobutyraldehyde. According our observations, replacement
The synthesis consisted of a free radical random copolymerization
of the co-monomers carried out in methanol at 60 C in a presence
◦
of DIPEA with a milder alkali, NaHCO , significantly lowered
of AIBN as the initiator (Scheme 4).
3
the amount of byproducts observed. This was probably due to
its low basicity and, therefore, low reactivity towards the 4-
iodobutyraldehyde (base catalyzed aldol condensation). The latter
reaction could be the source of undesired byproducts. The above
mentioned modification simplified the original procedure, as there
was no need to purify the crude product by preparative HPLC.
Syntheses of HPMA and MA-HH co-monomers, used for
the preparation of the reactive polymer precursor bearing the
hydrazide groups, were carried out as described previously (Hruby
et al., 2007; Ulbrich et al., 2000). Both the procedures were repro-
ducible; similarly, all characteristics of the products obtained were
in a good accordance with the literature.
The substance of the conversion of DOX to p-DOX is an alkyla-
tion of a primary amino group on the saccharide moiety of DOX.
Because of the substantially long distance between the amino and
carbonyl groups in the DOX molecule, the chemical reactivity of
the latter remains unchanged. This fact made possible the synthe-
sis of the p-DOX/polymer conjugate based on the same strategy as
that used for DOX, which was linked to a polymer via a hydrazone
bond derived from its carbonyl group. The respective polymer pre-
cursor was therefore synthesized by the same procedure that was
previously described (Etrych et al., 2008). Its chemical composition
corresponded to a copolymer of HPMA with MA-HH in an appro-
priate molar ratio with respect to a desired final content of p-DOX.
A different strategy than that used for DOX had to be developed
to attach the p-DOX to the polymer precursor. This was because an
N-alkylation of the polymer hydrazide groups with the protonated
pyrroline ring of p-DOX (see Scheme 1) under acidic conditions
necessary for successful condensation (acetic acid in methanol).
Consequently, an uncontrolled increase in molecular weight and
in the polydispersity of the formed PHPMA/p-DOX conjugate was
observed. Therefore, the transformation of DOX to p-DOX was
accomplished by derivation of DOX that was already attached to
a polymer. The reaction procedure consisted in a direct alkylation
of the previously prepared PHPMA/DOX conjugate with an excess
of 4-iodobutyraldehyde under mildly alkaline conditions (sodium
acetate) in methanol (Scheme 5). This technique represented an
effective, immediate way to synthesize the PHPMA/p-DOX conju-
gate with nearly quantitative conversion of DOX to p-DOX and no
recordable undesired side-reactions.
3.2. Inhibition of in vitro cell proliferation
Table 1 summarizes IC50 values (the drug concentration induc-
ing 50% cell death) that were obtained for the free p-DOX and
its PHPMA conjugate and for the doxorubicin and its PHPMA
conjugate. The data show similar cytostatic activities of p-DOX
and PHPMA/p-DOX, even in DOX resistant neuroblastoma cancer

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
161
DOX
poly(HPMA-co-MA-HH)
O
O
O
OH
CH3
CH3
OH
CH2
CH2
OH
O
O
a
b
NH
NH
+
O
OH
O
CH2
(CH2)
5
O
CHOH
O
CH3
NH
OH
NH2
NH2
acetic acid methanol
CH3
CH3
CH2
CH2
O
O
a
b
NH
NH
I
CH2
(CH2)
5
O
CHOH
O
PHPMA/DOX
CH3
NH
N
CH COONa
3
O
OH
OH
OH
OH
O
O
O
O
CH3
CH3
OH
NH2
CH2
CH2
O
a
O
b
NH
NH
CH2
(CH2)5
CH OH
CH3
O
NH
N
PHPMA/p-DOX
O
O
OH
OH
OH
OH
O
O
O
OH
N
Scheme 5. A two-step synthesis of PHPMA/p-DOX conjugate. a:b ∼ 98:2.
cell line UKF-NB4-DOXO. The PHPMA/p-DOX conjugate exhib-
ited 1.3- to 5-fold lower cytotoxicy, depending on the cancer cell
line, than free p-DOX. This is in agreement with the data already
obtained for DOX and its HPMA copolymer conjugates. A remark-
able difference, up to four orders of magnitude, could be seen
comparing the pharmacological efficacy of the HPMA copolymer
derivatives of doxorubicin and p-doxorubicin (UKF-NB4-DOXO cell
line).
3.3. Evaluation of in vivo acute toxicity
Acute systemic toxicities of free p-DOX and its polymer conju-
gate were assessed on the basis of changes in the body weight of
treated mice after drug administration. The data for various doses
are shown in Figs. 1 and 2.
Table 1
IC50 values in EL4 murine T cell lymphoma, 38C13 murine B cell lymphoma, UKF-NB4
neuroblastoma and its DOX resistant subline.
Our results show significantly suppressed toxicity of p-DOX
when attached to the polymer. Whereas a single dose of 1 mg/kg
of free p-DOX was lethal for the animals, the same dose of its poly-
mer form was only slightly toxic and non-lethal. These data are
in accordance with previous findings demonstrating a reduction
of toxicity of low-molecular-weight compounds after their attach-
ment to the polymer (Rihova, 2003). This suggests considerably
improved tolerance of the polymer form of drug in possible cancer
treatment.
Sample
IC50 concentration (ng/ml)a,b
EL4
0.07
0.38
7
38C13
UKF-NB4
UKF-NB4-DOXO
p-DOX
PHPMA/p-DOX
DOX
0.25
0.33
2.7
0.18
0.33
8
0.11
0.31
362
PHPMA/DOX
70
13
95
2 080
a
Related to the parent drug content.
Concentration which inhibits incorporation of [ H]thymidine by cells to 50%.
b
3

1
62
M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
23
22
21
20
19
18
17
16
15
0.6 mg/kg
1.2 mg/kg
100
1
.8 mg/kg
80
60
40
20
0
control
0.01 mg/kg
0.1 mg/kg
1 mg/kg
0
5
10
15
0
20
40
Day
60
80
Day
Fig. 1. Average weight of the mice after the treatment with a single i.v. dose of
p-DOX at day 0.
Fig. 4. Survival of mice bearing T-cell lymphoma EL4 treated with doses of 0.6, 1.2
and 1.8 mg p-DOX eq/kg of PHPMA/p-DOX conjugates. A single dose was adminis-
tered intravenously at day 9 after tumor inoculation.
2
2
2
2
2
1
1
1
1
1
1
4
3
2
1
0
9
8
7
6
5
4
to its polymer conjugates; therefore, the experiment was finished
at day 25.
The overall survival of mice bearing T-cell lymphoma EL4 (group
of 7) at three different doses of the PHPMA/p-DOX conjugate
(related to the parent drug content) is shown in Fig. 4.
A dose of 0.6 mg/kg represented a therapeutically efficient
amount of the conjugate, where 3 of 7 mice were cured with
complete tumor regression (Fig. 5). In contrast, the higher doses,
0
0
1
5
.01 mg/kg
.1 mg/kg
mg/kg
mg/kg
1
.2 mg/kg and 1.8 mg/kg, exhibited a critical toxicity where all
treated mice died by day 24 and day 16, respectively, while the
average survival of mice in the control group was 42 days. The
clearly fatal effect of these elevated doses can be attributed solely to
the systemic toxicity of p-DOX, as only a marginal development of
the average tumor volume in the respective animals was observed
0
5
10
Day
15
20
Fig. 2. Average weight of mice after the treatment with a single i.v. dose of
PHPMA/p-DOX conjugate at day 0. The doses are related to the parent drug.
(Fig. 5).
The first significant shrinkage of cancer was detectable in some
3
.4. Suppression of tumor growth in vivo
experimental animals on day 3 after the treatment. Mice that
did not respond by day 10 after the treatment were considered
treatment-resistant.
Changes inaverage body weightofmice in eachgroup areshown
in Fig. 6, and this information supports the acute toxicity of the
PHPMA/p-DOX conjugate. The data obtained showed an imme-
diate loss of average body weight of mice treated with doses of
The in vivo therapeutic effect of the PHPMA/p-DOX conjugate
was assessed on the EL4 T cell lymphoma-bearing C57BL/6 male
mice. Tumor growth, weight of animals and overall survival of ani-
mals were recorded.
Fig. 3 shows the initial tumor growth of mice bearing T-cell
lymphoma EL4 treated with the PHPMA/p-DOX conjugate and
with free p-DOX at approximately equitoxic doses. Whereas the
free p-DOX (0.1 mg/kg) exhibited no tumor growth suppression,
the PHPMA/p-DOX conjugate induced a significant retardation of
tumor development at doses of 0.4 and 0.8 mg eq/kg. The data
clearly showed that free p-DOX was completely inactive contrary
1
.2 and 1.8 mg p-DOX eq/kg, indicating their strong toxic effect. In
contrast, the dose of 0.6 mg/kg caused only non-significant loss of
body weight, peaking two weeks after drug administration, fol-
lowed by a course usual for undamaged animals. These findings
are in accordance with the above mentioned overall survival data,
thus indicating the dose 0.6 mg/kg as a therapeutical optimum for
mice bearing T-cell EL4 lymphoma.
8,000
6,000
4,000
2,000
0
free p-DOX
poly(HPMA)/p-DOX at 0.4 mg/kg
poly(HPMA)/p-DOX at 0.8 mg/kg
control
8,000
6,000
4,000
2,000
0
0.6 mg/kg
1.2 mg/kg
1.8 mg/kg
control
0
5
10
15
20
25
0
20
40
Day
60
80
Day
Fig. 3. Initial tumor growth of mice bearing T-cell lymphoma EL4 (group of 8)
treated with free p-DOX at dose 0.1 mg/kg and PHPMA/p-DOX conjugate at doses of
Fig. 5. Tumor growth in mice bearing T-cell lymphoma EL4 treated with doses 0.6,
1.2 and 1.8 mg drug eq/kg of PHPMA/p-DOX conjugate. A single dose was adminis-
tered intravenously at day 9 after tumor inoculation.
0
.4 and 0.8 mg p-DOX eq/kg. A single dose was administered intravenously at day 8
after tumor inoculation.

M. Studenovsky et al. / European Journal of Pharmaceutical Sciences 42 (2011) 156–163
163
3
3
2
2
1
1
5
0
5
0
5
0
drug, but its narrow “therapeutic window” emphasizes great care
required for the accurate dosage setting.
0
1
1
.6 mg/kg
.2 mg/kg
.8 mg/kg
Acknowledgements
control
Financial support from the Grant Agency of the Academy of Sci-
ences of the Czech Republic (grant no. IAAX 00500803) is gratefully
acknowledged. We also kindly thank Helena Misurcova and Pavla
Jungrova for maintaining tissue cultures, proliferation assays and
testing polymer drugs in vivo.
0
20
40
60
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