Synthesis and antitumor efficacy of a β-glucuronidase-responsive albumin-binding prodrug of doxorubicin

Article abstract of DOI:10.1021/jm300348r

In this paper we describe the synthesis and biological evaluation of the first β-glucuronidase-responsive albumin-binding prodrug designed for the selective delivery of doxorubicin at the tumor site. This prodrug leads to superior antitumor efficacy in mice compared to HMR 1826, a well-known glucuronide prodrug of doxorubicin that cannot bind covalently to circulating albumin. Furthermore, this compound inhibits tumor growth in a manner similar to that of doxorubicin while avoiding side effects induced by the free drug.

Full text of DOI:10.1021/jm300348r

Brief Article
pubs.acs.org/jmc
Synthesis and Antitumor Efficacy of a β-Glucuronidase-Responsive
Albumin-Binding Prodrug of Doxorubicin
Thibaut Legigan,† Jonathan Clarhaut,‡ Brigitte Renoux,† Isabelle Tranoy-Opalinski,† Arnaud Monvoisin,§
Jean-Marc Berjeaud,∥ Francois Guilhot,‡ and Sebastien Papot*,†
̧
́
†Institut de Chimie des Milieux et des Mater
86022 Poitiers, France
́ ́
iaux de Poitiers, IC2MP, Universite de Poitiers, UMR-CNRS 7285, 4 Rue Michel Brunet,
‡INSERM CIC 0802, CHU de Poitiers, 2 Rue de la Milet
́
rie, 86021 Poitiers, France
§Universite
́
de Poitiers, CNRS-FRE 3511, 1 Rue Georges Bonnet, 86022 Poitiers, France
∥
́
́
Ecologie et Biologie des Interactions, Equipe Microbiologie de l’Eau, Universite de Poitiers, UMR-CNRS 7267, 40 Avenue du
Recteur Pineau, 86022 Poitiers, France
S
* Supporting Information
ABSTRACT: In this paper we describe the synthesis and biological evaluation of the first β-glucuronidase-responsive albumin-
binding prodrug designed for the selective delivery of doxorubicin at the tumor site. This prodrug leads to superior antitumor
efficacy in mice compared to HMR 1826, a well-known glucuronide prodrug of doxorubicin that cannot bind covalently to
circulating albumin. Furthermore, this compound inhibits tumor growth in a manner similar to that of doxorubicin while avoiding
side effects induced by the free drug.
retention of macromolecular drug carriers in solid tumors,
which are too large to pass the endothelial barrier of healthy
tissues (the enhanced permeability and retention of macro-
molecules in solid tumors is known as the EPR effect6). Taking
advantage of the ERP effect, Kratz and co-workers developed
highly innovative albumin-binding prodrugs7 that demonstrated
remarkable antitumor activity.8 The originality of this targeting
strategy relies on the in vivo formation of a macromolecular
drug carrier resulting from the selective and rapid coupling of a
maleimide-containing prodrug with the cysteine 34 position of
circulating albumin after intravenous administration.8a In
addition to their discriminating accumulation in malignant
tissues, these macromolecules exhibit a favorable pharmacoki-
netic profile due to the prolonged half-life of albumin in the
body. Within this framework, INNO-206 (formerly DOXO-
EMCH), an albumin-binding prodrug of doxorubicin, is
currently being assessed clinically.9
In this paper, we report the synthesis and biological
evaluation of the first β-glucuronidase-responsive albumin-
binding prodrug 1 (Scheme 1). This compound includes a
glucuronide trigger, the potent doxorubicin, and a self-
immolative linker10 bearing a poly(ethylene glycol) side chain
terminated by a maleimide functional group.11 After intra-
venous administration, the in situ binding of prodrug 1 to the
thiol at the cysteine 34 position of plasmatic albumin via
Michael addition will produce the macromolecular drug carrier
2. Therefore, the larger size of the glucuronide 2 should prevent
the rapid renal clearance observed with β-glucuronidase-
responsive prodrugs developed so far. Once in targeted
tumor tissues, the β-glucuronidase-catalyzed cleavage of the
INTRODUCTION
■
Despite several years of intensive research devoted to the
discovery of novel anticancer agents, chemotherapy is still not
entirely effective for the treatment of many solid tumors. Most
of the drugs used clinically act by antiproliferative mechanisms
and lack any intrinsic selectivity, leading to severe adverse
effects due to the destruction of normal tissues. Therefore, the
development of more selective therapeutic approaches has
become a major goal in medicinal chemistry.
Over the past 2 decades, numerous glucuronide prodrugs
were investigated with the aim to deliver potent cytotoxic
agents exclusively in the vicinity of the tumor.1−3 Such enzyme-
responsive systems can be activated selectively by β-
glucuronidase located in high concentration in the micro-
environment of a wide range of solid tumors including lung,
breast, and gastrointestinal tract carcimomas.4 The validity of
this targeting strategy was demonstrated in mice with several
glucuronide prodrugs that showed superior antitumor efficacy
associated with reduced toxicity compared to standard
chemotherapy.5 However, the potential of this approach is
limited by the rapid elimination of glucuronide prodrugs by the
kidneys. As a result, the administration of very high doses is
usually required to achieve a significant therapeutic effect,
therefore representing a major problem for their clinical use.
Under these circumstances, the design of novel glucuronide
prodrugs exhibiting improved half-life is of interest to enhance
the efficiency of this promising drug delivery system.
In another approach, the passive targeting of tumor
anatomical features was also studied for the selective deposition
of anticancer agents in malignant tissues. Indeed, tumor
vasculature is characterized by tortuous, irregularly shaped
and leaky blood vessels. These anomalies combined with the
lack of lymphatic drainage allow the passive accumulation and
Received: March 13, 2012
Published: April 19, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Principle of Tumor Targeting with Maleimide-
Containing Glucuronide Prodrug 1
Scheme 2. Synthesis of Prodrug 1
a
Reagents and conditions: (i) doxorubicin, Et3N, HOBt, DMF, rt, 12
h, 79%; (ii) NH2CH2(CH2OCH2)10CH2N3, Cu(CH3CN)4PF6,
CH2Cl2, rt, 16 h; (iii) (CH3)2SO, rt, 16 h, 57% (two steps); (iv)
Pd(PPh3)4, aniline, THF/H2O (9:1), rt, 4 h, 40% after purification by
preparative chromatography.
catalyzed azide−alkyne 1,3-cycloaddition (CuAAC).13 After 16
h under these conditions, the reaction mixture was washed with
an aqueous solution of EDTA, the solvent was removed, and
the crude residue was engaged in the next step without any
further purification. In this case, reaction of the primary amine
of derivative 6 with the N-hydroxysuccinimide ester 7 afforded
the protected glucuronide 8 in 57% yield over two steps.
Finally, the expected prodrug 1 was obtained by the cleavage of
protecting groups performed with Pd(PPh3)4 as a catalyst and 2
equiv of aniline (40%, purity of >95% after purification by
preparative chromatography).
Biological Results. To demonstrate that the macro-
molecular drug carrier 2 can be formed spontaneously under
physiological conditions, prodrug 1 and human serum albumin
(HSA) were incubated together in phosphate buffer (pH 7) at
37 °C and the evolution of the mixture over time was
monitored by HPLC. As shown in Figure 1a, the peaks
corresponding to the two diastereoisomers of 1 disappeared
almost completely after 5 h of incubation. In contrast, when the
experiment was conducted with HMR 1826, a glucuronide
prodrug of doxorubicin that does not contain a maleimide
functional group (for the structure of HMR 1826 see
Supporting Information), the mixture appeared perfectly stable
with time (Figure 1b). As demonstrated previously by Kratz
and co-workers with other albumin binding prodrugs,8 this
indicated that prodrug 1 reacts with HSA through its
maleimide-containing side chain. Furthermore, since more
than 90% of 1 reacted with HSA in 2 h, it may be anticipated
that the kinetics of formation of the albumin conjugate 2 is
compatible with the plasmatic half-life of glucuronide prodrugs
such as HMR 1826.14 The subsequent addition of β-
glycosidic bond will trigger the release of doxorubicin in a
stringently controlled fashion through the self-immolative
mechanism depicted in Scheme 1. The results obtained in
this study show that prodrug 1 leads to a higher antitumor
efficacy than HMR 1826,12 a well-known glucuronide prodrug
of doxorubicin that cannot bind to circulating albumin.
Furthermore, this compound inhibits tumor growth in similar
a manner to that of doxorubicin while avoiding side effects
recorded with the free drug when administered at its maximal
tolerate dose.
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of prodrug 1 was carried out
starting from the fully allyl protected glucuronide 4 which is
readily accessible as a mixture of two diastereoisomers through
a multistep strategy recently described in the literature (Scheme
2).3d With this design, the cleavage of both allyl ester and
carbonates can be realized at the very end of the synthesis in a
one-step procedure under mild conditions that are compatible
with the presence of either alkali- or acid-sensitive moieties
such as doxorubicin or a maleimide. Thus, coupling between
the activated carbonate 4 and doxorubicin undertaken via
nucleophilic substitution in the presence of Et3N and HOBt
gave the alkyne 5 in 79% yield. The latter was then placed in
CH2Cl2 with commercially available O-(2-aminoethyl)-O′-(2-
azidoethyl)nonaethylene glycol and Cu(CH3CN)4PF6 in order
to form the triazole 6 through the well-known copper(I)-
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Journal of Medicinal Chemistry
Brief Article
Figure 1. Chromatograms of HSA incubated with prodrug 1 (a) or
HMR 1826 (b) in a 0.02 phosphate buffer (pH 7, 37 °C). β-
Glucuronidase was added at t = 5 h.
Figure 2. Tumor growth inhibition of subcutaneous murine LLC
xenografts under therapy with doxorubicin, HMR 1826, and prodrug 1
administered intravenously at days 4 and 11.
glucuronidase in the media (t = 5 h, Figure 1) resulted in the
clean release of doxorubicin which was fully completed 5 h after
the incubation of the activating enzyme (t = 10 h, Figure 1).
Taken together, these results demonstrated that prodrug 1
possesses the capacities to bind to HSA and to release the free
drug upon enzymatic activation.
Prodrug 1 was then evaluated for its antiproliferative activity
against human H290, MDA-MB-231, U87-MG, and murine
LLC tumor cell lines after 48 h of treatment (Table 1). When
As shown in Table 2, HMR 1826 was poorly active at the
tested dose (T/C = 69%). In contrast, prodrug 1 produced a
Table 2. Antitumor Activity of Doxorubicin, HMR 1826, and
Prodrug 1 against Murine LLC Xenografts in Vivo
dose
[μmol/kg]
body weight
a
T/C [%]
b
agent
mortality
change [%]
(day)
doxorubicin
HMR 1826
1
2 × 13.7
2 × 43.6
2 × 43.6
1/8
0/8
0/8
−21
+15
+8
31 (17)
69 (13)
24 (17)
Table 1. IC50 (nM) of Doxorubicin and Prodrug 1 with or
without β-Glucuronidase Determined by Cell Viability
a
b
a
Measured at day 17. T/C = [mean relative tumor volume of treated
Assays
group]/[mean relative tumor volume of control group]. As a
parameter for maximum efficacy, the minimal T/C was given on the
day it was obtained.
agent
H290
MDA-MB-231
U87-MG
LLC
doxorubicin
1
320 24
na
340 26
na
790 171
na
207 34
na
good antitumor response (T/C = 24%) which was comparable
to that obtained with doxorubicin (T/C = 31%). The higher
activity of 1 compared to HMR 1826 could be explained by the
formation of the macromolecular drug carrier 2 in the
bloodstream leading to enhanced permeability and retention
in the tumor combined with prolonged half-life. Moreover, the
free doxorubicin induced a body weight loss of 21%, whereas
prodrug 1 was well tolerated without any sign of overt toxicity.
As nephrotoxicity is one of the important side effects of
anthracycline antibiotics, histopathological examination of
kidneys was undertaken. This investigation revealed atrophy
of the renal pericapsular tissues in the group treated with
doxorubicin which correlates with the decreased body weight
observed clinically in these animals (Figure 3). On the other
hand, kidneys of mice that received iv injection of glucuronide
1 did not exhibit microscopic evidence of such toxicity. Overall,
these in vivo experiments demonstrated that the albumin-
binding prodrug 1 is a promising candidate for selective
treatment of solid tumors in vivo.
1 + β-Glu
310 62
320 65
850 34
173 36
a
na: no activity.
incubated alone, glucuronide 1 did not affect viability of cells at
the highest tested dose of 2 μM whereas the free drug was
highly toxic. This result indicated that derivatization of
doxorubicin in the form of prodrug 1 markedly reduced its
cytotoxicity. On the other hand, addition of β-glucuronidase in
the culture medium induced a dramatic antiproliferative effect
similar to that recorded for doxorubicin. As expected, enzymatic
activation of the glucuronide trigger led to the efficient release
of the drug, thereby restoring its initial activity. Further
experiments conducted on LLC cells showed that prodrug 1 is
4-fold less toxic than HMR 1826 in the absence of β-
glucuronidase (IC50 of 14 and 3.5 μM, respectively) while these
two targeting devices exhibit the same antiproliferative activities
when incubated with the activating enzyme (IC50 of 173 and
199 nM, respectively). This finding suggests that the additional
hydrophilicity imparted by the polyethylene glycol-containing
side chain limits passive cellular uptake and further intracellular
activation of 1 by lysosomal β-glucuronidase.
The in vivo efficacy of prodrug 1 was assessed in a
subcutaneous murine Lewis lung carcinoma (LLC) implanted
in C57BL/6 mice. The animals received two iv injections of
43.6 μmol/kg of prodrug 1 or HMR 1826 at days 4 and 11 after
transplantation. Doxorubicin was tested at its maximal tolerate
dose (2 × 13.7 μmol/kg) following the same therapeutic
protocol (Figure 2).
Figure 3. Sections of the kidney stained with hematoxylin eosin: (a)
untreated; (b) treated with doxorubicin; (c) treated with prodrug 1.
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Brief Article
13.95 (2s, 1H). 13C NMR (100 MHz, CDCl3) δ 17.0, 25.2, 26.5, 28.4,
29.8, 30.2, 32.9, 34.0, 35.7, 36.4, 37.8, 39.2, 47.3, 50.2, 53.5, 56.8, 65.6,
66.9, 67.4, 67.7, 68.6, 69.2, 69.3, 69.4, 69.5, 69.7, 70.0, 70.3, 70.4−70.7,
72.2, 72.4, 73.6, 73.9 and 74.0, 75.1, 76.7, 77.9, 99.5, 100.9, 111.4,
111.6, 118.6, 119.1, 119.3, 119.4, 119.5, 120.0, 121.0, 122.9, 123.2,
123.9, 130.9, 131.0, 131.1, 131.3, 132.1, 132.5, 133.7, 134.2, 135.6,
135.9, 140.7, 140.9, 142.4, 148.7, 153.5, 153.6, 154.0, 154.6, 154.7,
155.7, 155.8, 156.3, 161.2, 165.6, 165.7, 170.9, 172.9, 186.8, 187.2,
214.0. HRMS (ESI) [M + Na]+ m/z 1986.7184 (calcd for
C91H117N7O41Na, 1986.71777), [M + 2Na]2+ m/z 1004.8535 (calcd
for C91H117N7O41Na2, 1004.8535), [M + Na + K]2+ m/z 1012.8404
(calcd for C91H117N7O41NaK, 1012.840 47).
Synthesis of Prodrug 1. To a solution of 8 (166 mg, 0.084
mmol) in THF/H2O, 9/1 (2 mL), were added Pd(PPh3)4 (14.6 mg,
15 mol %) and aniline (19 μL, 0.211 mmol). Total deprotection was
achieved after stirring at room temperature for 4 h. Solvents were
removed under reduced pressure. High degree of purity for 1 was
obtained using preparative-reverse phase HPLC (56 mg, 40%, purity
>95%). Mp = 140−150 °C (dec). 1H NMR (400 MHz, CDCl3) δ 1.14
(m, 5H), 1.46 (m, 4H), 1.83 (m, 1H), 2.01−2.19 (m, 4H), 2.94 (m,
2H), 3.14−3.75 (m, 49H, partially masked by H2O residual peak),
3.88−3.99 (m, 4H), 4.12 (m, 1H), 4.37−4.44 (m, 2H), 4.55 (s, 2H),
4.91 (m, 1H), 5.17−5.47 (m, 4H), 5.76 (m, 1H), 7.0 (s, 2H), 7.10 and
7.23 (2s, 1H), 7.32 (m, 1H), 7.47−7.55 (m, 1H), 7.66 (m, 1H), 7.75−
7.83 (m, 2H), 7.91 (m, 2H), 13.26 (1s, 1H), 14.05 (1s, 1H). 13C NMR
(100 MHz, CDCl3) δ 17.0, 24.8, 25.8, 27.8, 32.11, 35.1, 37.0, 38.3 and
38.4, 47.1, 49.3, 52.3, 54.9, 56.6, 63.7, 66.7, 67.9, 68.8, 69.2, 69.6−69.9,
71.1, 72.7, 74.9, 75.3, 75.8, 99.8, 100.1, 110.7, 110.8, 112.0, 115.7,
115.8, 116.5 and 116.8, 117.7, 118.6, 118.8, 119.0, 119.8, 120.0, 122.7,
123.5 and 123.6, 128.9, 130.2, 130.3, 132.0, 133.7 and 133.8, 134.1,
134.4, 134.7, 135.5, 136.3, 139.5, 139.7, 141,8 and 141,9, 148.5, 154.4
and 154.5, 156.0 and 156.1, 157.6, 157.9, 160.8, 170.0, 171.1, 172.0,
186,5 and 186.6, 213.8. HRMS (ESI) [M − H]− m/z 1670.6264 (calcd
for C76H100N7O35, 1670.626 58).
CONCLUSION
■
In summary, we prepared a glucuronide prodrug of doxorubicin
bearing a maleimide-containing side chain which allows its
binding to plasmatic albumin through Michael addition under
physiological conditions. This β-glucuronidase-responsive
targeting system is more efficient than its analogue HMR
1826 when administrated at the same dose in mice.
Furthermore, our albumin-binding prodrug exhibits an
antitumor efficacy similar to that of doxorubicin while avoiding
side effects induced by the free drug. This finding could be of
interest in the search for efficient anticancer prodrug for
selective chemotherapy of solid tumors.
EXPERIMENTAL SECTION
■
General Method. 1H and 13C NMR spectra were recorded at 400
MHz at ambient temperature in the indicated solvent. High-resolution
ESI mass spectra were obtained using a Waters MicrO-Tof-Q2 mass
spectrometer. Analytical thin layer chromatography was performed
using 0.2 mm silica gel 60-F plates. Flash chromatography was carried
out with silica gel 60 (15−40 μm) as the stationary phase. Purity of
compound 1 was determined to be >95% by HPLC.
Synthesis of 5. To a solution of doxorubicin hydrochloride (207
mg, 0.36 mmol) in DMF (3.8 mL) was added Et3N (49 μL, 0.36
mmol). The mixture was stirred at room temperature for 30 min.
HOBt (48 mg, 0.36 mmol) and a solution of 4 (300 mg, 0.36 mmol)
in DMF (2.5 mL) were added. The mixture was stirred at room
temperature for 7 h and concentrated in vacuo. The crude product was
purified by column chromatography over silica gel (CH2Cl2/MeOH,
99/1, 98/2) to afford 5 (364 mg, 83%) as a mixture of two
diastereoisomers (red solid). Mp = 140−150 °C (dec). 1H NMR (400
MHz, CDCl3) δ 1.27 (m, 3H), 1.81 (m, 3H), 1.97 (m, 1H), 2.09−2.51
(m, 4H), 2.66 (m, 2H), 2.91 (m, 1H), 3.19 (m, 1H), 3.59 (s, 0.5H),
3.67 (s, 0.5H), 3.81 (m, 1H), 4.09 (m, 4H), 4.30 (m, 1H), 4.56−4.72
(m, 10H), 5.21−5.36 (m, 13H), 5.48 (m, 1H), 5.65 (m, 1H), 5.80−
5.93 (m, 4H), 7.27 (m, 2H), 7.37 (m, 1H), 7.47 (m, 1H), 7.77 (m,
2H), 7.98 (m, 1H), 13.15 (2s, 1H), 13.91 (2s, 1H). 13C NMR (100
MHz, CDCl3) δ 16.9, 26.4 and 26.5, 30.1 and 30.2, 34.0, 35.7, 47.2 and
47.3, 56.8, 65.6, 67.0, 67.4, 69.3, 69.4, 69.5, 69.6 and 69.7, 69.8, 71.9
and 72.0, 72.3, 72.4, 72.5 and 72.6, 73.9 and 74.0, 75.0 and 75.1, 76.7,
78.6, 99.4 and 99.6, 100.7 and 100.8, 111.4, 111.6, 118.6 and 118.7,
118.8, 119.1, 119.4, 119.5, 119.9, 120.8, 123.4, 123.6, 130.9, 131.0,
131.1, 131.3, 132.3 and 132.4, 133.5 and 133.6, 133.6 and 133.7, 135.4
and 135.5, 135.9, 140.6 and 140.8, 149.0, 153.5 and 153.6, 154.0,
154.3, 155.6, 156.2, 161.1, 165.6 and 165.7, 186.6 and 186.7, 187.0 and
187.1, 213.9. HRMS (ESI) [M + Na]+ m/z 1267.3222 (calcd for
C59H60N2O28Na, 1267.32248), [M + K]+ 1283.3005 (calcd for
C59H60N2O28K, 1283.296 42).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Structure of HMR 1826, H NMR and 13C NMR spectra of 5,
8, and 1, HPLC conditions, cell viability assays, curves
depicting body weight change. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +33 549 453 862. E-mail: sebastien.papot@univ-
poitiers.fr.
■
Notes
The authors declare no competing financial interest.
Synthesis of 8. To a solution of 5 (186 mg, 0.149 mmol) and O-
(2-aminoethyl)-O′-(2-azidoethyl)nonaethylene glycol (102 mg, 0.149
mmol) in CH2Cl2 (7 mL) was added Cu(MeCN)4PF6 (78 mg, 0.209
mmol). The mixture was stirred at room temperature for 3 h, and a
solution of disodium EDTA (956 mg, 2.568 mmol) in 0,1 M
phosphate buffer (14 mL) was added. The resulting mixture was
stirred for 5 h and extracted with CH2Cl2 (3 × 50 mL). The combined
organic layers were dried over MgSO4 and concentrated in vacuo. The
crude product was dissolved in DMSO (3 mL), and 7 (58 mg, 0.194
mmol) was added. The mixture was stirred at room temperature for 16
h, and CH2Cl2 (50 mL) was added. The organic layer was washed with
water (5 × 50 mL), dried over MgSO4, and concentrated in vacuo.
The crude product was purified by column chromatography over silica
gel (CH2Cl2/MeOH, 95/5, 92/8) to afford 8 (166 mg, 57%) as a
mixture of two diastereoisomers (red solid). Mp = 140−150 °C (dec).
1H NMR (400 MHz, CDCl3) δ 1.21−1.33 (m, 5H), 1.54−1.76 (m,
5H), 1.82−1.90 (m, 1H), 2.15 (m, 3H), 2.31 (d, 1H, J = 14.1 Hz),
2.98 (m, 1H), 3.22 (m, 3H), 3.44−3.82 (m, 46H), 4.07 (m, 4H), 4.30
(m, 1H), 4.47−4.73 (m, 12H), 5.19−5.36 (m, 13H), 5.48 (m, 1H),
5.73−6.06 (m, 5H), 6.26 (bs, 1H), 6.69 (s, 2H), 7.27 (m, 2H), 7.41
(m, 2H), 7.67 (m, 2H), 7.78 (m, 1H), 8.02 (m, 1H), 13.22 (2s, 1H),
ACKNOWLEDGMENTS
The authors thank CNRS and La Ligue Nationale contre le
■
Cancer (Comite
this study.
́
Charente-Maritime) for financial support of
ABBREVIATIONS USED
■
EPR, enhanced permeability and retention; rt, room temper-
ature; HOBt, N-hydroxybenzotriazole; THF, tetrahydrofuran;
DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide;
EDTA, ethylenediaminetetraacetic acid; HSA, human serum
albumin; β-Glu, β-glucuronidase; iv, intravenous
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