Fine tuning of the pH-dependent drug release rate from polyHPMA- ellipticinium conjugates

Article abstract of DOI:10.1016/j.bmc.2013.07.038

Polymer conjugates of anticancer drugs have shown high potential for assisting in cancer treatments. The pH-labile spacers allow site-specific triggered release of the drugs. We synthesized and characterized model drug conjugates with hydrazide bond-containing poly[N-(2-hydroxypropyl) methacrylamide] differing in the chemical surrounding of the hydrazone bond-containing spacer to find structure-drug release rate relationships. The conjugate selected for further studies shows negligible drug release in a pH 7.4 buffer but released 50% of the ellipticinium drug within 24 h in a pH 5.0 phosphate saline buffer. The ellipticinium drug retained the antiproliferative activity of the ellipticine.

Full text of DOI:10.1016/j.bmc.2013.07.038

Accepted Manuscript
Fine tuning of the pH-dependent drug release rate from polyHPMA-ellipticini‐
um conjugates
Ondřej Sedláček, Martin Studenovsky, David Větvička, Karel Ulbrich, Martin
Hrubý
PII:
S0968-0896(13)00659-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.07.038
BMC 11002
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 May 2013
17 July 2013
19 July 2013
Please cite this article as: Sedláček, O., Studenovsky, M., Větvička, D., Ulbrich, K., Hrubý, M., Fine tuning of the
pH-dependent drug release rate from polyHPMA-ellipticinium conjugates, Bioorganic & Medicinal Chemistry
(2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.07.038
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Graphical Abstract
Fine Tuning of the pH-Dependent Drug
Release Rate from PolyHPMA-Ellipticinium
Conjugates
Leave this area blank for abstract info.
Ondrej Sedlacek, Martin Studenovsky, David Vetvicka, Karel Ulbrich, Martin Hruby
Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Fine tuning of the pH-dependent drug release rate from polyHPMA-ellipticinium
conjugates
Ondřej Sedláčeka, Martin Studenovskya, David Větvičkab, Karel Ulbricha, Martin Hrubý a*1
a Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
b Institute of Biophysics and Informatics, First Faculty of Medicine, Charles University in Prague, Salmovska 1, 120 00 Prague 2, Czech Republic
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Polymer conjugates of anticancer drugs have shown high potential for assisting in cancer
treatments. The pH-labile spacers allow site-specific triggered release of the drugs. We
synthesized and characterized model drug conjugates with hydrazide bond-containing poly[N-
(2-hydroxypropyl)methacrylamide] differing in the chemical surrounding of the hydrazone
bond-containing spacer to find structure-drug release rate relationships. The conjugate selected
for further studies shows negligible drug release in a pH 7.4 buffer but released 50% of the
ellipticinium drug within 24 h in a pH 5.0 phosphate saline buffer. The ellipticinium drug
retained the antiproliferative activity of the ellipticine.
Available online
Keywords:
ellipticine
controlled release
hydrazone
2009 Elsevier Ltd. All rights reserved.
polyHPMA
drug delivery
1. Introduction
ligand-based targeting.25, 26 The conjugate releases its cargo in a
biologically active form in the tissues or cells of tumors. A
Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is an
isoquinoline-type alkaloid that naturally occurs in the Ochrosia
elliptica plant.1 Ellipticine shows a strong antineoplastic effect
based mainly on the DNA intercalation and topoisomerase II
inhibition.2-6 However, the low water solubility of ellipticine
prevented it from entering clinical trials.7 Therefore, its water-
soluble derivatives with a quaternary ammonium structural motif
were synthesized.8 These include elliptinium (Celiptium®, i.e., 2-
methyl-9-hydroxyellipticine),9, 10 methoxycelliptium (9-methoxy-
2-methyl-ellipticine)11 datelliptium [2(diethylamino)ethyl-9-
hydroxyellipticine],12 retelliptine (1-diethyl-aminopropylamino-
suitable linker between the drug and the carrier is essential for
such a delivery system.27 This linker must be stable during the
transport through the blood and must rapidly hydrolyze to release
the drug in the tumor, triggered by various factors such as a pH
change after crossing from the blood into the tumor or tumor cell
environments.1
A hydrazone bond was first used as a part of the spacer
connecting doxorubicin to a polymer carrier,28-30 and it quickly
became popular for the synthesis of polymer conjugates with pH-
controlled drug release.31 It is relatively stable in pH of blood
(7.4), thus allowing the derivative to reach the tumor tissue
without a substantial chemical change. However, in the mildly
acidic environment of the tumor interstitial space (pH variable
around 6.5 depending on actual oxygen supply and metabolic
activity in each part of the tumor) or even in the pH of late
endosomes (as low as pH 5.0), the drug cargo rapidly releases
9-methoxyellipticine),13
elliprabin
(-arabinosyl-9-
15
hydroxyellipticine)14,
or the bis-ellipticinium derivative
ditercalinium.15 From these, elliptinium and datelliptium have
been used for the treatment of advanced breast cancer.9 However,
severe side effects, including nephrotoxicity,16 renal toxicity,
hemolysis,17 xerostomia, nausea and vomiting,18 have been
observed.
33
from the polymer.32, The release profile of the drug can be
strongly influenced by variations in the chemical structure of the
spacer in the vicinity of the hydrazone bond, and it can be fine-
tuned for the delivery of each drug.34, 35 Due to this and because
ellipticine lacks the oxo group needed for conjugation with
hydrazine or the hydrazide group-bearing polymer carrier,
functionalization of the drug with suitable oxo group-containing
linker is necessary. However, when modifying it, one must take
care not to hamper the biological activity of the original drug
The targeted delivery and controlled release using water-
soluble polymer carriers became a widely studied approach to
improve the solubility and circumvent the side effects of
anticancer drugs.19-22 The drug is attached to a polymer carrier.
This biologically inactive prodrug conjugate is delivered to the
cancer tissue, where it accumulates through e.g., Enhanced
23, 24
of macromolecules or
P—er—me—ation and Retention (EPR)
*Corresponding author, Tel: +420 296 809 230, Fax: +420 296 809 410, E-mail: mhruby@centrum.cz

because it will then be released in its substituted form. To
connect ellipticine to the polymer, the drug may be converted to
its derivative by quaternization of the ellipticines’ isoquinoline-
type nitrogen using a suitable linker, as previously described by
our group.36 A higher affinity between these derivatives and
DNA (due to the permanent positive charge in the molecule) also
benefits this function.37
To assess the influence of steric hindrance on the cleavage of
acidic hydrazone, the isoquinolinium conjugates containing
methyl (2a), ethyl (2b), isopropyl (2c) and tert-butyl (2d) groups
adjacent to the ketone were synthesized, and their hydrolytic
release profiles were determined. In the phosphate buffer that
was used to model the pH of blood (pH 7.4), nearly no low
molecular weight isoquinolinium models were released in any
instance. This could be explained by the strong electron-
withdrawing effect of the permanent positive charge in the beta
position to the ketone. The release of the drug was significantly
faster when exposed to a pH of 5.0, which simulated the pH in
late endosomes. Sterical hindrance had a dramatic effect on the
release rate, the polymer conjugate of the methyl derivative 2a
had the fastest release rate, and the polymer conjugate of tert-
butyl derivative 2d had the slowest release rate. This slow release
rate could be ascribed to the steric hindrance of the transition
state, which is most likely to have hybridization close to the sp2
state.38
In this paper, we describe the effect of the structural changes
of the linker that is used to connect the ellipticinium drug with
the polymer carrier on the pH-dependent release profile of the
drug. Understanding these structural changes will allow us to
fine-tune the ellipticinium drug delivery systems. To the best of
our knowledge, no such study on ellipticine derivatives has been
published so far, and the only study on ellipticinium hydrazone
conjugates was our paper on the multilevel targeting of an Auger
electron emitter 125I.36
2. Materials and Methods
To determine the influence of adjacent permanent positive
charge on the rate of hydrolysis, we compared the release of the
aforementioned derivative 1a, which contained a positive charge
in the -position respective to the oxo-group, with the derivatives
with positive charges in the - (1e) and - (1f), respectively -
(1f) positions, from their conjugates. It can be clearly observed in
Figures 2 and 3 that the presence of a positive charge proximal
to the original ketone substantially reduces its release rate. This
decrease of release rate made the derivative with the closest
charge (-oxo derivative 2a) the most stable derivative, whereas
the conjugate with most remote charge (-oxo derivative 2f) was
the most labile conjugate, even at a pH of 7.4 (77 % of the drug
released within 24 h). The release rate of the latter two
conjugates (i.e., 2e-f) is comparable with the release rates
obtained with common aliphatic linkers (e.g., levulinic acid)
studied for the conjugation of drugs to polymers via the
Detailed desctiption of the materials, characterization of
products and experimental procedures could be found in
Supplementary Material.
3. Results and Discussion
Isoquinoline was used as a cheap and relatively non-toxic
model for the release profile screening of ellipticine. This
compound was quaternized readily with plethora bromo- or
tosyloxy ketones to produce oxo-alkyl isoquinolinium salts 1a-f.
These were conjugated with hydrazide groups-containing HPMA
copolymer (Mw = 24.5 kDa, Mw/Mn = 1.87) by acetic acid-
catalyzed condensation,33 forming derivatives 2 (Figure 1). The
molecular weights and polydispersity indexes of all conjugates
confirmed no cross-linking of chains, and the content of
isoquinolinium salts was 0.8 – 5.6% wt. See supporting materials
for detailed experimental procedures and characterization data.
hydrazone
bond.34
X
Y
Z-
a
b
-CH2-
-CH2-
-Me
-Et
Br
Br
2a-f
c
d
e
f
-CH2-
-iPr
-tBu
-Me
-Me
Br
1a-f
-CH2-
Br
-(CH2)2-
-(CH2)3-
TsO-
Br-
5
4
=
3
Figure 1. Syntheses and structures of the polymer conjugates.

This behavior could be explained by the electrostatic
disinclination of hydrazones with proximate positive charges
towards their protonation as the first step of the hydrolysis
mechanism. A similar explanation was also used in the case of
the hydrolysis of charged acetals.39
conjugate with the oxobutyl-linker described in our previous
work.36
To assure the DNA-intercalation ability, the solution of
ellipticinium derivative 4 was titrated with the solution of calf-
thymus DNA, and the fluorescence of the mixture was
measured.40 As a result, the fluorescence emission of the solution
gradually rises with the addition of DNA, which confirms
intercalation. The blue shift in fluorescence emission maximum
is also consistent with more hydrophobic microenvironment of
the intercalated molecule compared to free molecule in aqueous
solution. The DNA affinity constant of the ellipticinium 4 and of
ellipticine was determined using a Scatchard plot.41 As described
previously, the ellipticine derivatives exhibit two different
binding modes depending on the drug/DNA ratio.42 At a low
drug/DNA ratio, ellipticinium derivative 4 binds with the
intercalation constant K=2.17x107 M-1(bp) (see Table 1 and
supplementary information), with an average of one molecule of
ellipticine to 4.57 DNA base pairs (bp), which is in the same
range as the ellipticine standard (K=3.81x107 M-1(bp) and a ratio
of 5.42 bp per molecule of the drug). Lower (0.57-times) stability
of the complex of derivative 4 with DNA compared to similar
complex with protonated ellipticine may be explained by sterical
reasons on the quaternary amine, which is sterically more
demanding than just protonated ellipticinium.
100
1a pH 5.0
1b pH 5.0
1c pH 5.0
80
1d pH 5.0
60
40
20
0
0
12
24
36
48
t (h)
Figure 2. The release profile of derivatives 1a-d from their
conjugates 2a-d in phosphate buffered media at 37°C. The
release in the pH 7.4 phosphate buffer was under 2% after 24 h.
100
4 pH 5.0
4 pH 7.4
80
60
40
20
0
100
80
1e pH 7.4
1e pH 5.0
1f pH 7.4
1f pH 5.0
60
40
20
0
0
12
24
t (h)
36
48
Figure 4. The release profile of derivative 4 from conjugate 5
in phosphate buffer media at 37°C.
At high drug/DNA ratios, the second binding mode occurs,
and the higher intercalation density of 2.18 bp per molecule of
the ellipticinium 4, as well as the lower intercalation constant
K=6.12x105 M-1(bp), are observed (for the ellipticine standard,
the values K=5.9x105 M-1(bp) and a ratio of 2.65 bp/drug were
determined). All values are consistent with those described in the
literature for similar compounds and thus confirm the retention of
the intercalation ability of 4.42
0
12
24
36
48
t (h)
Figure 3. The release profile of derivatives 1e-f from their
conjugates 2e-f in phosphate buffer media at 37°C.
Of all the linkers described above, the simplest 2-oxopropyl
linker showed the best release profile for cancer applications
(negligible at pH 7.4 and sufficiently fast in a slightly acidic
milieu) and was thus chosen to connect ellipticine to the
hydrazide-containing pHPMA polymer in the same manner as
described for the model isoquinolinium derivatives above. The
drug release profile of the modified ellipticinium polymer
conjugate (5) was determined (Figure 4). At a pH of 7.4,
conjugate 5 has shown remarkable stability, and only a negligible
amount of ellipticinium 4 was released. At a pH of 5.0, over 50%
of the drug was released from the polymer within 24 h. This
slightly slower release rate than that of the conjugate 2a can be
explained by the enhanced electron delocalization of hydrazones’
-electrons in the aromatic system of the ellipticinium in
comparison with isoquinolinium. However, the release of the
drug is still more than two-fold faster than in the case of the
Furthermore, we tested the antiproliferative activity of 2-N-(2-
oxopropyl)ellipticinium bromide (5) on selected cell lines (4T1,
Raji and EL4, respectively).34 Cytotoxicities, expressed as the
IC50 values, were in the range of 2.7 - 7.1 mol/L for 4, whereas
those of ellipticine were in the range of 1.0 - 8.3mol/L (Table
1). The EL4 cell line was the most sensitive, and the 4T1cell line
was the least sensitive to both compounds. The differences
between the IC50 values for different cell lines were statistically
significant (analysis of variance, ANOVA on the level α = 0.05)
for both ellipticine and 4. However, the IC50 values were not
statistically significantly different when comparing ellipticine
and 4 for any cell line tested (analysis of variance, ANOVA on
the level α = 0.05). One can thus conclude that ellipticine and its
low molecular weight derivative 4 possess equal antiproliferative

IC50b, mol/L
Raji
Compound
4
K [107 M-1(bp)]a
2.17 ± 0.29
4T1
EL4
7.1 ± 0.8
8.3 ± 0.8
2.8 ± 0.7
7.7 ± 1.1
2.7 ± 0.5
1.0 ± 0.2
ellipticine
3.81 ± 0.62
Table 1. DNA binding and antiproliferative activity of ellipticinium derivative 4 and ellipticine. a DNA-intercalation constant obtained
from the compound fluorescence changes upon the addition of CT-DNA (K ± standard deviation). b Concentrations caused a 50%
inhibition in the MTT test (IC50 ± standard deviation, n = 5) in mol/L.
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released from its conjugates without side reactions. The data on
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the in vitro cytotoxicity of the conjugates may thus be misleading
due to the significantly different concentrations of the drug
released into the media during incubation with the cells compared
with the in vivo situation.30 This is because in an in vivo situation,
the system is opened, i.e., the released drug is being continuously
removed by internalization into cells or diffusion out of the tumor
tissue. In addition, the pH of tumor tissue is generally slightly
acidic but varies according to the exact location in the tumor by 1
– 1.5 pH units, which has a dramatic effect on the drug release
rate and therefore the published IC50 values of hydrazone
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We have shown that ellipticinium derivatives with
antiproliferative activity can be bound to a polymer carrier by a
hydrolytically labile bond with a widely tunable release rate. The
key structural features that determine the release rate are
proximity of the positive charge and the sterical hindrance. The
optimized derivative showed no less antiproliferative activity
when compared with ellipticine. The optimized derivative also
exhibited a negligible release rate at pH modeling blood plasma
(pH 7.4) and a sufficient release rate in an environment that
modeled pH in late endosomes (pH 5.0; 50 % drug released
within 24 h of incubation). The rules found for the described
system have the potential to aid further designs of biodegradable
spacers for biomedicinal applications also in other drug delivery
systems.
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The financial support from the Grant Agency of the Czech
Republic (grant # P304/12/0950), from the Ministry of Industry
and Trade of the Czech Republic (grant # MPO TIP FR-TI4/625)
and from the Academy of Sciences of the Czech Republic (grant
# M200501201) is gratefully acknowledged.
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*Graphical Abstract (for review)