Development of enzymatically cleavable prodrugs derived from dendritic polyglycerol

Article abstract of DOI:10.1016/j.bmcl.2009.05.058

In this Letter we report the synthesis and in vitro studies of cleavable polymer-drug conjugates derived from dendritic polyglycerol and maleimide-bearing prodrugs of doxorubicin and methotrexate that are cleaved by cathepsin B. Cleavage properties and cytotoxicity of the new conjugates are presented.

Full text of DOI:10.1016/j.bmcl.2009.05.058

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3725–3728
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of enzymatically cleavable prodrugs derived from dendritic
polyglycerol
a,
Marcelo Calderón a, Ralph Graeser b, Felix Kratz c, , Rainer Haag
*
*
a Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
b Proquinase, Breisacher Strasse 117, 79106 Freiburg, Germany
c Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter we report the synthesis and in vitro studies of cleavable polymer–drug conjugates derived
from dendritic polyglycerol and maleimide-bearing prodrugs of doxorubicin and methotrexate that are
cleaved by cathepsin B. Cleavage properties and cytotoxicity of the new conjugates are presented.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 16 February 2009
Revised 13 May 2009
Accepted 14 May 2009
Available online 18 May 2009
Keywords:
Polyglycerol
Polymer therapeutics
Prodrug
Doxorubicin
Methotrexate
Drug conjugate
Controlled release
Nanocarrier
Many active molecules used in biomedical applications, for
example, anticancer drugs, imaging probes, genes, etc., present a
lack of specificity due to their low molecular weights which results
in a uniform biodistribution, low concentration at the site of action,
rapid plasma clearance, and high potential toxicity. These limita-
tions can be overcome by the design of new macromolecular car-
rier systems. Several strategies have been pursued, that include
active and passive targeting approaches with antibodies, serum
proteins, liposomes, and synthetic polymers.1–4
Combination of active molecules with polymers may reduce
their toxicity, eliminate undesirable body interactions, improve
their solubility, bioavailability, stability, and prolong blood clear-
ance.5 Moreover, suitable polymer-based drug delivery systems
can enable controlled release and specific delivery of bioactive
agents to the diseased or damaged tissue (by non-covalent or cova-
lent attachments).
ene glycol (PEG), and poly(glutamic acid).6 In addition, albumin, as
a biopolymeric carrier, has been evaluated as a drug delivery sys-
tem in anticancer therapy.7
In the design of new macromolecular carrier systems there is a
great need for polymers that are highly water-soluble, nontoxic,
nonimmunogenic, and biodegradable with low polydispersity and
well-defined functional groups.8 In this regard, dendritic polymers
possess unique features that may be advantageous for drug deliv-
ery.9–12 They can be tailored on a defined nano-sized scale with
low polydispersity and present a high functionality which allows
the conjugation of multiple units of drugs, targeting moieties, sol-
ubilizing agents as well as agents which could reduce potential
toxicity. The loading ability, water solubility, biodistribution prop-
erties, and therapeutic efficacy can be easily tuned by varying
structural properties such as the core, branching units, surface
groups, or the size of the dendritic core.
Although significant efforts are being made to develop novel
polymeric carriers, synthetic polymers which have been used in
clinically evaluated drug conjugates are mainly restricted to N-
(2-hydroxypropyl) methacrylamide copolymer (HPMA), polyethyl-
In recent years many studies were published using dendritic
polymers in gene and drug delivery, magnetic resonance, and fluo-
rescence imaging. Several examples of dendritic molecules have
been introduced for biomedical applications such as polyamido-
amine,10 polylysine,13 polyester,14,15 polyglycerol,16–19 and tria-
zines dendrimers.20
* Corresponding authors. Tel.: +49 761 2062930; fax: +49 761 2062905 (F.K.),
tel.: +49 30 838 52633; fax: +49 30 838 53357 (R.H.).
E-mail addresses: felix@tumorbio.unifreiburg.de (F. Kratz), haag@chemie.fu-
berlin.de (R. Haag).
Chemical conjugation to a dendritic scaffold, thereby exploiting
the high multivalency, allows for the covalent attachment of differ-
ent kinds of active molecules (imaging agents, drugs, targeting
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.058

3726
M. Calderón et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3725–3728
moieties, or biocompatible molecules) in a controlled ratio. The
loading as well as the release can be tuned by incorporating cleav-
able bonds which can be degraded under the specific conditions
present at the site of action (e.g., acidic pH, over-expression of spe-
cific enzymes or reductive media).
The covalent attachment of drug molecules to polymers is a
promising route for better controlling the loading and release of
active molecules as compared to physical encapsulation. Therefore,
there is a need for systems where the conjugation and the release
of drugs are strictly controlled using biodegradable dendritic
scaffolds.21
Here we present the synthesis of a scaffold derived from hyper-
branched polyglycerol (PG, Fig. 1) and its subsequent conjugation
with enzymatically cleavable prodrugs of doxorubicin and metho-
trexate (Fig. 2),22,23 and report on the release behavior as well as
the cytotoxicity of the new drug polymer conjugates against two
human tumor cell lines.
The increasing development of maleimide-bearing prodrugs
and diagnostic dyes23,24 instigated us to synthesize thiolated nano-
carriers with tuneable properties such as molecular weight, solu-
bility, or targeting potential by selective functionalization of
polyglycerol hydroxyl groups.25 Therefore, we developed a strategy
to synthesize thiolated hyperbranched polyglycerols that can be
used as a general, flexible method to couple diagnostic or thera-
peutic agents under physiological conditions.
The synthetic protocol consisted of four steps. The first three
steps, shown in Scheme 1, were carried out as reported earlier
for the synthesis of polyglycerolamine with an average molecular
weight of 20 kDa and 20% of total hydroxyl groups functionalized
to amine groups.26 For the synthesis of the thiolated derivatives,
three different pathways were studied using 3-(tritylthio)propi-
onic acid (A), 2-iminothiolane (B), or acetyl-thiopropionic acid
(C). In each case the optimal conditions for synthesis and purifica-
tion were studied as a function of the reaction time, solvent, stoi-
chiometry and purification method using UV–vis and 1H NMR
spectroscopy (see Supplementary data).
Among all the thiolation processes studied, the 2-iminothiolane
pathway (B) was the most reproducible for the in situ Michael
reaction with maleimide derivatives as the following step. The
thiol formation over time was confirmed by 1H NMR and Ellman’s
test. After 5, 15, 30, 60, 90, 120, 150, 180, 270, 330, and 1220 min
samples were taken from the reaction mixture and evaluated. A
signal shift from 3.21 to 2.58 ppm in the 1H NMR spectra corre-
sponding to the methylene group in position 2 from 2-iminothio-
lane as well as the absorbance at 412 nm in UV–vis after
incubation with 5,50-dithio-bis(2-nitrobenzoic acid) was used for
monitoring the degree of thiolation. A maximum in the degree of
thiolation was reached after 40 min. Standardization of this step
HO
HO
OH
O
HO
O
OH
OH
OH
O
OH
HO
HO
OH
OH
OH
O
O
HO
HO
O
OH
O
O
O
HO
OH
OH
O
O
OH
O
O
OH
O
O
HO
O
OH
OH
O
O
OH
OH
OH
OH
O
O
OH
OH
O
OH
O
OH
O
O
O
O
OH
HO
O
HO
OH
O
O
HO
O
OH
O
O
OH
OH
O
HO
O
OH
OH
O
O
O
O
OH
O
O
OH
HO
O
OH
OH
OH
O
HO
O
HO
O
O
O
O
HO
O
O
HO
OH
OH
O
O
O
OH
OH
O
O
O
OH
HO
OH
O
O
HO
HO
O
O
O
O
O
O
O
OH
OH
O
O
OH
HO
HO
O
O
O
HO
O
OH
OH
HO
O
O
HO
O
O
HO
O
OH OH
O
O
O
O
O
O
HO
OH
HO
O
O
O
O
HO
HO
O
OH HO
O
HO
OH
O
O
HO
O
OH
O
O
HO
HO
O
OH
HO
O
O
OH
O
O
OH
HO
O
O
O
O OH
OH
O
OH
O
HO
HO
HO
O
O
HO
O
HO
O
HO
O
O
OH
HO
HO
HO
O
HO
O
OH
O
O
O
HO
O
O
O
HO
HO
O
HO
HO
O
O
O
HO
HO
O
HO OH
HO
HO
HO
HO
O
O
HO
O
HO
HO
HO
HO
HO
HO
PG
OH
OH
Figure 1. Schematic representation of hyperbranched polyglycerol (PG).
O
OH
OH
O
O
a
HO
HO
O
O
O
O
O
O
O
CH C
CH2
CH2
CH2
CH2
NH2
N
H
H
N
O
N
CH C
CH2
N
H
O
O
N
H
OH
CF3COOH
O
b
O
O
CH C
CH3
O
O
O
H
H
H
H
N
N
N
CH C
CH2
N
CH C
CH2
CH2
CH2
CH2
NH2
N
CH C OH
CH2
O
CH2
CH2
HO
O
CH2
O
HN
N
H
NH2
O
N
CF3COOH
N
N
N
N
NH2
Figure 2. Chemical structures of (a) EMC-Phe-Lys-DOXO, and (b) EMC-D-Ala-Phe-Lys-Lys-MTX (EMC = e-maleimidocaproic acid).

M. Calderón et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3725–3728
3727
NaN3
MsCl
PPh3
OH
N
3
OMs
PG
NH
2
PG
PG
PG
DMF
60° C, 72 h
Pyridine
0° C - r.t., 18 h
THF/Water
r.t., 24 h
OH
OH
OH
OH
O
O
O
O
N
O
S
1.
(A)
(B)
O
N
H
SH
PG
NH
PG
2
2. NH2OH
OH
OH
O
DRUG
N
S
NH
NH Cl
2
2
NH
2
O
O
N
PG
SH
S
N
H
PG
N
H
DRUG
OH
PBS pH 7
r.t., 1 h
O
O
N
O
O
O
S
Enzymatically
cleavable bond
O
1.
N
H
SH
PG
(C)
OH
2. TFA/Tri-isopropylsilane
Scheme 1. Studied pathways for synthesis of thiolated polyglycerol and prodrug coupling.
allowed a convenient control over the amount of thiols generated
for further drug conjugation that was performed as a one pot reac-
tion in aqueous systems.
For conjugation, we used a maleimide-bearing prodrug of doxo-
rubicin or methotrexate which incorporate either a self-immola-
tive para-aminobenzyloxycarbonyl (PABC) spacer coupled to the
550
500
450
400
350
300
250
200
150
100
50
Free doxorubicin
dipeptide Phe-Lys or the tripeptide D-Ala-Phe-Lys as the protease
substrate (see Fig. 2). Both prodrugs have been shown to selec-
tively bind to the cysteine-34 position of human serum albumin,
they are cleaved by cathepsin B, an enzyme overexpressed by sev-
eral solid tumors,27 to release doxorubicin or a methotrexate lysine
derivative and they exhibit superior antitumor efficacy in vivo over
the free drug.22,23
2.5 h
5 min
0
0 h
-50
0
10
20
30
40
50
The conjugation between thiolated polyglycerol and the pro-
drugs was achieved through selective Michael addition between
the maleimide group of the prodrugs and the sulfhydryl groups
from thiolated polyglycerol in PBS of pH 7. The thiol group adds
to the double bound of the maleimide group in a fast and selective
reaction at room temperature forming a stable thioether bond.
Highly water soluble conjugates, PG-Phe-Lys-DOXO and PG-D-
Ala-Phe-Lys-Lys-MTX, were obtained with this protocol with pay-
loads of 45% and 23% wt/wt for doxorubicin and methotrexate,
respectively.30
T [min]
Figure 3. Size-exclusion chromatogrammes of an incubation study of PG-Phe-Lys-
DOXO at pH 5.0 and 37 °C with cathepsin B. The cleavage product doxorubicin
elutes at ꢀ20 min.
An alternate uptake of the drug in its macromolecular form, fol-
lowed by intracellular cleavage, might explain the improved activ-
ity observed. In any case, however, the conjugates are active in the
low micromolar range that is relevant for further preclinical stud-
ies of anticancer prodrugs.
Subsequently, drug release of the conjugates was studied in the
presence of cathepsin B using size exclusion HPLC.31 An effective
cleavage of PG-Phe-Lys-DOXO and PG-D-Ala-Phe-Lys-Lys-MTX
and release of doxorubicin and methotrexate-lysine in presence
of the enzyme was observed. As an example, the chromato-
grammes for the time-dependent release of doxorubicin are shown
in Figure 3 that demonstrate that doxorubicin is liberated over
time.
The antiproliferative activity of doxorubicin, methotrexate and
the PG drug conjugates was assessed against two human tumor
cell lines, AsPC1 LN (pancreatic carcinoma) and MDA-MB-231 LN
(mamma carcinoma), which were both transfected with the lucif-
erase gene using a luciferase assay (see Table 1).32 Whereas in
the case of doxorubicin, the IC50 values for the PG drug conjugates
were lower than for the free drugs, the methotrexate conjugates
appeared more active than the free drug. MDA-MB231 cells have
been shown to be deficient in methotrexate uptake.28
Table 1
IC50 values of doxorubicin, methotrexate, PG-Phe-Lys-DOXO, and PG-D-Ala-Phe-Lys-
Lys-MTX
Compounds
MDA-MB 231 IC50
SD]
AsPC1 IC50
SD]
[l
M
[lM
Doxorubicin
PG-Phe-Lys-DOXO
Methotrexate
0.13 0.06
1.10 0.4
0.26 0.09
2.4 0.6
>100
lM
>100
lM
PG-D-Ala-Phe-Lys-Lys-MTX
13.30 0.9
8.9 1.8
For IC50 measurements, 0.2 ꢁ 104 cells were plated per well in a 96-well plate and
serial dilutions of the drugs were added in triplicates. After 72 h cells were lysed in
100 lL of luciferase assay buffer, and 10 lL of the lysate were assayed for luciferase
activity. Methotrexate treatment did not reduce the cell proliferation rate below
50% during the experiment.

3728
M. Calderón et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3725–3728
26. Roller, S.; Zhou, H.; Haag, R. Mol. Diversity 2005, 9, 305.
In summary, we have developed a new macromolecular nano-
27. Jedeszko, C.; Sloane, B. F. Biol. Chem. 2004, 385, 1017.
carrier from a dendritic polyglycerol scaffold. Optimal conditions
of synthesis and purification were studied for three different inter-
mediates pathways. Selective conjugation with maleimide-bearing
prodrugs as well as drug release in the presence of cathepsin B has
been successfully demonstrated. Cytotoxicity of the conjugates
against human tumor cell lines showed that the activity of the
drugs was primarily retained which encouraged us to study the
physical properties and use these systems for further in vivo
studies.
28. Worm, J.; Kirkin, A. F.; Dzhandzhugazyan, K. N.; Guldberg, P. J. Biol. Chem. 2001,
276, 39990.
29. Haag, R.; Mecking, S.; Türk, H. Patent Application DE10211664A1, 2002.
30. Note 1. General procedure for prodrug coupling: Polyglycerol (Mn = 20,000 g/mol,
PD = 2.0) was prepared according to published procedures.29 Polyglycerol
amine (DF = 20%) was prepared as previously described.21 The conjugation
reactions were performed at room temperature with vigorous stirring for
80 min. To two different flasks, containing 10 mL of
a solution of
polyglycerolamine (10 mg/mL, 2.7 mmol NH2-groups) in sterile 5 mM EDTA,
50 mM PBS solution pH 7.0 were added 4.5 mL of a solution of 2-iminothiolane
(2 mg/mL in the same solvent system, 1.2 equiv). After 20 min, prodrug
solutions were added (3 mg/mL in 5%
D-(+)-glucose pH 3.0 for EMC-Phe-Lys-
Doxo and 3.7 mg/mL in PBS solution for EMC-
D
-Ala-Phe-Lys-Lys-MTX). The
Acknowledgments
total amounts of each prodrug were added in three repeated aliquots until the
first evidence of precipitation was apparent, waiting ten minutes for addition
of each aliquot. After 1 h of reaction, the solutions were concentrated to 5 mL
using Centriprep YM10 (twice at 4000 U/min for 30 min). Polyglycerol-drug
conjugates were purified by gel-filtration through a Sephadex G-25 column
(Amersham) with PBS solution (pH 7) yielding approximately 10–15 mL of a
red and yellow solution, respectively. A second concentration with Centriprep
was made and finally the conjugates were lyophilized to yield a red (for
doxorubicin conjugate) or yellow (for methotrexate conjugate) crystalline
powder.Conjugate formation was studied by chromatography on reverse phase
TLC, by appearance of a faster band in sephadex column, and by SEC-HPLC.
Absence of physical encapsulation was investigated by performing the same
coupling procedure with polyglycerolamine alone. The drug concentration of
We thank the Ministry of Science for their continuing support of
this work for a NanoFuture award for R. Haag and F. Kratz (BMBF
03X5501).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.058.
the
conjugates
495 = 10645 Mꢂ1 cmꢂ1
370 = 7420 Mꢂ1 cmꢂ1
was
determined
photometrically
at
495 nm
(
(
e
e
)
for the doxorubicin conjugate and at 370 nm
for methotrexate conjugate, after reconstitution of
References and notes
)
the lyophilized conjugates in PBS solution pH 5.8.
31. Note 2. Cleavage studies of dPG drug conjugates with cathepsin B: Enzymatically
active cathepsin B was purchased from Calbiochem (Bad Soden, FRG). Stock
1. Haag, R.; Kratz, F. Angew. Chem., Int. Ed. 2006, 45, 1198.
2. Duncan, R. Nat. Rev. Cancer 2006, 6, 688–701.
3. Vicent, M. V.; Dieudonné, L.; Carbajo, R. J.; Pineda-Lucena, A. Expert Opin. Drug
Delivery 2008, 5, 593.
solution of the dPG drug conjugates (3000
lM) were mixed with cathepsin B
(71.7 g/mL, 110 mU) in 20 mM sodium acetate buffer pH 5.0, containing L-
l
4. Kiick, K. L. Science 2007, 317, 1182.
cysteine (8 mM). The samples were incubated at 37 °C and were collected over
16 h and analyzed by HPLC: column: BioSil SEC 250 from Biorad, mobile phase:
4 mM sodium phosphate, 0.15 M NaCl, 10% acetonitrile (pH 7.4), flow: 1.0 mL/
min; Kontron 422 pump and a Kontron 430 detector (at 495 and 220 nm). For
peak integration Geminyx software (version 1.91 by Goebel Instrumentelle
Analytik) was used. Control studies were carried out without cathepsin B
addition and no release was observed after 16 h of incubation.
ˇ
ˇ
ˇ
ˇ
5. Chytil, P.; Etrych, T.; Konák, C.; Šírová, M.; Mrkvan, T.; Boucek, J.; Ríhová, B.;
Ulbrich, K. J. Controlled Release 2008, 127, 121.
6. Kratz, F.; Abu Ajaj, K.; Warnecke, A. Expert Opin. Invest. Drugs 2007, 16, 1037.
7. Kratz, F. J. Controlled Release 2008, 132, 171.
8. Hespe, W.; Meier, A. M.; Blankwater, Y. J. Arzneim.-Forsch. 1977, 27, 1158.
9. Liu, M. J.; Fréchet, J. M. J. Pharm. Sci. Technol. Today 1999, 2, 393.
10. Lee, C. C.; MacKay, J. A.; Fréchet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005, 23,
1517.
11. Tomalia, D. A.; Reyna, L. A.; Svenson, S. Biochem. Soc. Trans. 2007, 35, 61.
12. Svenson, S. Eur. J. Pharm. Biopharm. 2009, 71, 445.
13. Sadler, K.; Tam, J. P. J. Biotechnol. 2002, 90, 195.
14. Ihre, H.; Padilla de Jesus, O. L.; Szoka, F. C. J.; Fréchet, J. M. J. Bioconjugate Chem.
2002, 13, 433.
32. Note 3. In vitro cellular experiments: Cytotoxicity with the dPG drug conjugates
and free drugs were performed with two human tumor cell lines: AsPC1 LN
(pancreatic carcinoma) and MDA-MB-231 LN (mamma carcinoma) both
transduced with the luciferase gene. Cells were grown as monolayer cultures
in cell culture flasks in DMEM Glutamax I culture medium supplemented with
10% heat-inactivated FCS, 100 U/mL penicillin, and 100 lg/mL streptomycin.
Cells were cultured in a humidified atmosphere of 95% air and 10% carbon
dioxide at 37 °C. Media were routinely changed every 3 days. For subculture or
experiments, cells growing as monolayer cultures were released from the
tissue flasks by treatment with 0.05% trypsine/EDTA. For the experiments, cells
were used during the logarithmic growth phase.For the cytotoxicity assay, cells
were plated at 200,000 cells/well into a 96-well plate. Stock solutions of the
dPG drug conjugates (3 mM) were diluted with DMEM culture medium to
15. Grinstaff, M. W. Chemistry 2002, 8, 2839.
16. Haag, R.; Sunder, A.; Stumbé, J. F. J. Am. Chem. Soc. 2000, 122, 2954.
17. Xu, S.; Luo, Y.; Graeser, R.; Warnecke, A.; Kratz, F.; Hauff, P.; Licha, K.; Haag, R.
Bioorg. Med. Chem. Lett. 2009, 19, 1030.
18. Frey, H.; Haag, R. J. Biotechnol. 2002, 90, 257.
19. Boysen, M. M. K.; Elsner, K.; Sperling, O.; Lindhorst, T. K. Eur. J. Org. Chem. 2003,
4376.
20. Neerman, M. F.; Zhang, W.; Parrish, A. R.; Simanek, E. E. Int. J. Pharm. 2004, 281,
129.
21. Khandare, J. J.; Minko, T. Prog. Polym. Sci. 2006, 31, 359.
22. Abu Ajaj, K.; Graeser, R.; Fichtner, I.; Kratz, F. Cancer Chemother. Pharmacol.
2009, 64, 413.
23. Warnecke, A.; Fichtner, I.; Sass, G.; Kratz, F. Arch. Pharm. 2007, 340, 389.
24. Tyagarajan, K.; Pretzer, E.; Wiktorowicz, J. E. Electrophoresis 2003, 24, 2348.
25. Kratz, F.; Müller, I. A.; Ryppa, C.; Warnecke, A. ChemMedChem 2008, 3, 20.
0.01–10
in 90 L of luciferase assay buffer (25 mM TRIS-phosphate pH 7.8; 2 mM EDTA;
2 mM DTT; 0.1% Triton X-100), and 10 L of the undiluted, as well as 1/10 and
lM and exposed to the cells for 72 h in triplicate. Cells were then lysed
l
l
1/100 dilutions were measured in a Luminometer (BMG Lumistar) using the
luciferase substrate from Promega (Promega E4550), according to the
manufacturer’s instructions. IC50 values were determined using GRAPHPAD
prism software.