The first generation of β-galactosidase-responsive prodrugs designed for the selective treatment of solid tumors in prodrug monotherapy

Article abstract of DOI:10.1002/anie.201204935

Massive attack: Galactoside prodrugs have been designed that can be selectively activated by lysosomal β-galactosidase located inside cancer cells expressing a specific tumor-associated receptor. This efficient enzymatic process triggers a potent cytotoxic effect, releasing the potent antimitotic agent MMAE and allowing the destruction of both receptor-positive and surrounding receptor-negative tumor cells. Copyright

Full text of DOI:10.1002/anie.201204935

Angewandte
Chemie
DOI: 10.1002/anie.201204935
Tumor Targeting
The First Generation of b-Galactosidase-Responsive Prodrugs
Designed for the Selective Treatment of Solid Tumors in Prodrug
Monotherapy**
Thibaut Legigan, Jonathan Clarhaut, Isabelle Tranoy-Opalinski, Arnaud Monvoisin,
Brigitte Renoux, Mikaꢀl Thomas, Alain Le Pape, Stꢁphanie Lerondel, and Sꢁbastien Papot*
In memory of Gꢁrard Dꢁlꢁris
The selective killing of tumor cells without affecting normal
tissues is one of the main challenges of cancer chemotherapy.
In recent years, the development of drug carriers designed to
deliver potent cytotoxic compounds exclusively inside malig-
nant cells has emerged as a valuable alternative to avoid dose-
limiting adverse effects recorded with traditional anticancer
agents.[1] Within this framework, the use of antibody–drug
conjugates[2] targeting specific tumor-associated antigens is by
far the best-explored approach. Many of these compounds are
currently evaluated in humans, including Brentuximab Vedo-
tin,[3] which reached the market in 2011 for the treatment of
lymphomas. Another promising strategy relies on the use of
nontoxic prodrugs that can be activated by an enzyme[4]
previously targeted in cancerous tissues by the mean of
a monoclonal antibody in the course of antibody-directed
enzyme prodrug therapy (ADEPT).[5] In this case, the active
compound is released extracellularly in the vicinity of cancer
cells that are subsequently killed after drug uptake. Over the
past decade, several galactoside prodrugs[6] have appeared as
potential candidates to achieve selective chemotherapy of
solid tumors in combination with antibody-b-galactosidase
conjugates.[7] The best illustration of such an enzyme-respon-
sive system is unambiguously the galactoside prodrugs of
duocarmycin analogues developed by Tietze and co-work-
ers[8] that meet all the main criterions required to be used in
ADEPT.[9] However, the implementation of ADEPT proce-
dures remains complex, costly, and with high risk of immune
response linked to the administration of an antibody–enzyme
conjugate. This is probably the reason why none of these
promising galactoside prodrugs has been assessed for anti-
tumor efficacy in animal models to date. Under such circum-
stances, the development of a simpler approach, allowing the
selective activation of galactoside prodrugs by the endoge-
nous b-galactosidase located inside malignant cells, is of great
interest.
Herein, we present the first generation of galactoside
prodrugs suitable for the treatment of solid tumors in prodrug
monotherapy (PMT[10]), a strategy in which the use of an
antibody-b-galactosidase is not needed. For this purpose, we
designed the novel drug delivery system 1 composed of
a galactoside trigger, a targeting ligand, and a potent cytotoxic
compound articulated around a central self-immolative linker
(Figure 1).[11]
[*] T. Legigan, Dr. I. Tranoy-Opalinski, Dr. B. Renoux, Dr. M. Thomas,
Dr. S. Papot
Universitꢀ de Poitiers, UMR-CNRS 7285, Institut de Chimie des
Milieux et des Matꢀriaux de Poitiers (IC2MP), Groupe “Systꢁmes
Molꢀculaires Programmꢀs”
4 rue Michel Brunet, 86022 Poitiers (France)
E-mail: sebastien.papot@univ-poitiers.fr
Dr. J. Clarhaut
INSERM CIC 0802, CHU de Poitiers
2 rue de la Milꢀterie, 86021 Poitiers (France)
Dr. A. Monvoisin
Universitꢀ de Poitiers, CNRS-FRE 3511
1 rue Georges Bonnet, 86022 Poitiers (France)
Dr. A. Le Pape, Dr. S. Lerondel
UPS n844 TAAM—CIPA, CNRS,
3B rue de la Fꢀrollerie, 45071 Orlꢀans (France)
[**] The authors thank the CNRS, La Ligue Nationale contre le Cancer
(Comitꢀs Charente, Charente-Maritime, Vienne, Deux-Sꢁvres),
Assoiciation Cent pour Sang la Vie, and the Cancꢀropꢂle Grand
Ouest for financial support of this study.
Figure 1. The principle of tumor targeting. Step 1: selective recognition
of receptor-positive cancer cells; step 2: receptor-mediated endocyto-
sis; step 3: b-galactosidase-catalyzed drug release; step 4: diffusion of
the drug into the nucleus or the cytoplasm of both receptor-positive
and receptor-negative cancer cells, leading to the death of each type of
cell.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201204935.
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Such a targeting assembly is programmed to be selectively
activated by the b-galactosidase present in the lysosomal
compartment of cancer cells expressing a specific tumor-
associated receptor.[1] Thus, as shown in Figure 1, recognition
of the membrane receptor by the targeting ligand (step 1) will
trigger the receptor-mediated endocytosis[12] of the whole
device (step 2) that will be followed by the intracellular
enzyme-catalyzed mechanism of drug release (step 3). As b-
galactosidase is present in lysosomes of both healthy and
malignant cells, this highly specific internalization process will
allow the prodrug activation to occur exclusively inside
receptor-positive tumor cells, thereby avoiding unselective
drug release in non-malignant tissues. However, as the
prodrug activation is catalytic, the b-galactosidase confined
in the targeted cells will trigger the liberation of sufficient
drug quantities to induce the death of both receptor-positive
and surrounding receptor-negative tumor cells (step 4). It is
worth mentioning that numerous tumor-associated recep-
tors[1] have already been identified, and consequently this new
generation of galactoside prodrugs could be adapted to target
a wide variety of malignancies.
two diastereoisomers through a multistep strategy that has
been already described (Scheme 2).[16] Indeed, as we previ-
ously demonstrated with other enzyme-responsive systems,[17]
glycosylated intermediates such as 2 are ideal platforms for
Scheme 2. Synthesis of the galactoside prodrug 1. a) MMAE, diisopro-
pylethylamine (DIPEA), hydroxybenzotriazole (HOBt), pyridine/DMF,
RT, 36 h, 75%; b) LiOH, MeOH, 08C, 20 min, 98%; c) 5, CuSO4,
sodium ascorbate, DMSO, RT, 8 h, 85%.
the successive introduction of a cytotoxic compound on the
activated carbonate at the benzylic position and a targeting
entity on the terminal alkyne of the linker unit. Thus, coupling
between 2 and MMAE undertaken by nucleophilic substitu-
tion gave the protected galactoside 3 in 75% yield. Full
deprotection of the hydroxy groups furnished the clickable
derivative 4 in nearly quantitative yield. Finally, introduction
of the folate ligand was conducted in the presence of the azide
5, using the well-known copper(I)-catalyzed azide–alkyne 1,3-
cycloaddition to afford the prodrug 1, which was then purified
by preparative chromatography for biological evaluations
(85%, as a mixture of four isomers).
As proof of the concept, we developed the pilot prodrug
1 that can be activated selectively inside folate receptor-
expressing tumor cells (Scheme 1). The folate receptor
We first investigated the ability of the prodrug 1 to target
selectively FR-positive tumor cells by measuring its antipro-
liferative activity against both KB and HeLa cells, which
overexpress the FR at various levels, as well as FR-negative
A549 cells. As shown in Table 1, our enzyme-responsive
Table 1: IC50 values (nm) of MMAE and prodrug 1 on KB, HeLa, and A549
cell lines correlated with the FR level.[a]
IC50 [nm]
Cell line
FR level
MMAE
1
KB
HeLa
A549
+++
++
À
0.240
0.630
0.872
0.240
8.408
195.230
Scheme 1. Structure of prodrug 1, and the b-galactosidase-catalyzed
MMAE release mechanism.
[a] Values represent the mean ÆSEM of seven experiments performed in
triplicate. KB cells: human mouth epidermal carcinoma; HeLa cells:
human cervix adenocarcinoma; A549 cells: human bronchial carcinoma.
FR: folate receptor.
(FR)[13] is indeed overexpressed in several cancer types[14]
while it is mainly undetectable in most normal tissues. Thus,
the FR-expressing cells represent targets of choice to
demonstrate the validity of this concept. With our design,
the b-galactosidase-catalyzed cleavage of the glycosidic bond
will release the potent antimitotic agent monomethyl auri-
statin E (MMAE)[15] in a stringently controlled fashion by the
self-immolative mechanism depicted in Scheme 1.
system dramatically affected the viability of KB and HeLa
cells, with IC50 values of 0.240 and 8.408 nm, respectively. The
cytotoxicities recorded in these experiments were consistent
with the FR expression level that is higher in KB than in
HeLa cells (see the Supporting Information). Interestingly,
the antiproliferative activity of the galactoside prodrug 1 in
KB cells is similar to that of MMAE (IC50 = 0.240 nm),
making this compound the most potent folate–drug conju-
The synthesis of prodrug 1 was carried out starting from
the galactoside 2, which is readily accessible as a mixture of
2
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gate[18] developed to date. On the other hand, A549 cells that
present only low level of FR were much less sensitive to the
incubation of 1 (IC50 = 195.230 nm), whereas MMAE was
highly toxic (IC50 = 0.872 nm). As expected, the hydrophilicity
imparted by the galactoside trigger prevented passive cellular
uptake and further intracellular activation of the prodrug in
non-targeted cells. All together, these results indicated that
the galactoside 1 can be selectively activated inside FR-
positive tumor cells. Thus, as most normal tissues express low
level of FR, such outcomes suggest that prodrug 1 should
present only reduced toxicity toward safe tissues compared to
untargeted MMAE in vivo.
KB FR-positive with A549 FR-negative cells using TransWell
Boyden chambers (Figure 3).
Prodrug 1 was incubated with KB cells placed in the top
chamber to trigger the release of MMAE, which can
To confirm that this selective toxicity was the consequence
of the intracellular activation of prodrug 1 by lysosomal b-
galactosidase, we examined the inhibition of tubulin poly-
merization, the mechanism by which MMAE exerts its
antitumor activity (Figure 2). Thus, as shown by confocal
Figure 3. Viability of A549 cells in co-culture assay using TransWell
Boyden chambers: a) in red, top chamber contains KB cells and
bottom chamber contains A549 cells; b) in green, both top and
bottom chambers contain A549; in blue, the viability of A549 cells
alone was used as a control.
subsequently diffuse through the 0.4 mm filter in the bottom
chamber containing A549 cells (Figure 3a). Under these
conditions, the targeting system 1 induced a dramatic anti-
proliferative effect on A549 cells, which was comparable to
that measured with MMAE at identical doses (5 and 10 nm).
As a negative control, the same experiments were conducted
with A549 cells in the top chamber (Figure 3b). In this case,
while the antimitotic drug affected the viability of cells,
prodrug 1 did not exhibit any significant toxicity. In accord-
ance with the principle of tumor targeting illustrated in
Figure 1, these results demonstrated for the first time that the
activation of a galactoside prodrug such as 1 by lysosomal b-
galactosidase located inside FR-expressing cells is an efficient
catalytic process, enabling the release of suitable quantities of
MMAE for the destruction of surrounding cancer cells,
whatever their membrane characteristics.
The in vivo efficacy of the galactoside prodrug 1 in the
course of PMT was assessed in nude mice bearing luciferase-
transfected KB xenografts. The animals received several
intravenous injections of 5 mgkgÀ1 of the prodrug starting at
day 5 after tumor implantation (for the full therapeutic
procedure, see the Supporting information). Tumor progres-
sion was monitored by bioluminescence imaging three times
per week and compared to that of mice treated with
0.1 mgkgÀ1 of MMAE (Figure 4a).
Figure 2. a-Tubulin immunodetection by confocal microscopy in HeLa
cells treated for 24 h with a) DMSO, b) MMAE at 1 nm, and c) prodrug
1 at 1 nm. White arrows indicate cells blocked by MMAE or 1.
microscopy imaging, incubation of the free MMAE with
HeLa cells disturbed the microtubule network (Figure 2b),
while this was not detected when cells were untreated
(Figure 2a). Furthermore, as demonstrated by FACS analysis
(see the Supporting Information), galactoside prodrug 1 pro-
duced a similar effect on cell division, demonstrating that its
selective receptor-mediated endocytosis is followed by the b-
galactosidase-catalyzed release of the antimitotic agent
MMAE (Figure 2c). The role of lysosomal b-galactosidase
in the prodrug activation process was also evidenced by
comparing a galactoside conjugate of doxorubicin with its
glucuronide analogue (see the Supporting Information).
As cancerous tissues are highly heterogeneous, the
selective destruction of a particular population of malignant
cells, such as those expressing a membrane receptor, is not
sufficient to eradicate the wide diversity of tumor cells.
However, an efficient intracellular enzymatic activation of
prodrug 1 should release high quantities of MMAE that could
then diffuse out of FR-positive cells to kill surrounding FR-
negative cancer cells. To verify this hypothesis, we co-cultured
As illustrated in Figure 4b, prodrug 1 induced a marked
antitumor activity with almost total and durable disappear-
ance of the luminescence from day 21, while treatment with
MMAE led only to a moderate inhibition of tumor growth.
With the aim of increasing the efficacy of the antimitotic
agent in this animal model, MMAE was also evaluated at
a higher dose of 0.5 mgkgÀ1. However, in this case the first
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Figure 5. Survival curve representing the percent survival as a function
of time in day.
prodrug 1 produces a remarkable antitumor effect when
tested against FR-expressing KB xenograft without any
detectable toxicity, showing the validity of this concept
in vivo. Furthermore, as the synthetic strategy employed for
the preparation of 1 allows the custom design of other b-
galactosidase-responsive targeting assemblies, this approach
could be easily adapted for the treatment of particular
malignancies based on their tumor-associated membrane
specificities. This new generation of low-molecular-weight
galactoside prodrugs may offer a valuable alternative to the
use of antibodies (in the form of either antibody-drug or
antibody-enzyme conjugates) that exhibit poor tumor pene-
tration. Thus, our finding may open a new door for selective
chemotherapy of solid tumors.
Received: June 24, 2012
Revised: July 16, 2012
Published online: && &&, &&&&
Figure 4. a) Representative bioluminescence imaging of luciferase-
transfected KB xenografts at days 5, 12, 17, and 21 post-implantation
when treated with vehicle (5% DMSO in PBS buffer), MMAE (intra-
venous administration at days 4, 7, 10, 14, and 17) or prodrug
1 (intravenous administration at days 5, 7, 10, 12, 14, 17, 19, 21, 23,
and 26); b) Tumor growth inhibition over the time under therapy with
prodrug 1 and MMAE.
Keywords: auristatin · cancer · galactoside · prodrugs ·
.
self-immolative linkers
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4
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Communications
Tumor Targeting
Massive attack: Galactoside prodrugs
have been designed that can be selec-
tively activated by lysosomal b-galactosi-
dase located inside cancer cells express-
ing a specific tumor-associated receptor.
This efficient enzymatic process triggers
a potent cytotoxic effect, releasing the
potent antimitotic agent MMAE and
allowing the destruction of both receptor-
positive and surrounding receptor-nega-
tive tumor cells.
T. Legigan, J. Clarhaut,
I. Tranoy-Opalinski, A. Monvoisin,
B. Renoux, M. Thomas, A. Le Pape,
S. Lerondel, S. Papot*
&&&& — &&&&
The First Generation of b-Galactosidase-
Responsive Prodrugs Designed for the
Selective Treatment of Solid Tumors in
Prodrug Monotherapy
6
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
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