Bioactivation of Self-Immolative Dendritic Prodrugs by Catalytic Antibody 38C2

Article abstract of DOI:10.1021/ja039052p

Self-immolative dendrimers have recently been developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units, through a self-immolative chain fragmentation, which is initiated b

Full text of DOI:10.1021/ja039052p

Published on Web 01/27/2004
Bioactivation of Self-Immolative Dendritic Prodrugs by
Catalytic Antibody 38C2
Marina Shamis,† Holger N. Lode,‡ and Doron Shabat*,†
Contribution from the School of Chemistry, Raymond and BeVerly Sackler Faculty of Exact
Sciences, Tel-AViV UniVersity, Tel AViV 69978, Israel, and Charite´ Children’s Hospital,
Humboldt UniVersity, Augustenburger Platz 1, 13353 Berlin, Germany
Received October 14, 2003; E-mail: chdoron@post.tau.ac.il
Abstract: Self-immolative dendrimers have recently been developed and introduced as a potential platform
for a multi-prodrug. These unique structural dendrimers can release all of their tail units, through a self-
immolative chain fragmentation, which is initiated by a single cleavage at the dendrimer’s core. Incorporation
of drug molecules as the tail units and an enzyme substrate as the trigger can generate a multi-prodrug
unit that will be activated with a single enzymatic cleavage. We have synthesized the first generation of
dendritic prodrugs with doxorubicin and camptothecin as tail units and a retro-aldol retro-Michael focal
trigger, which can be cleaved by catalytic antibody 38C2. The bioactivation of the dendritic prodrugs was
evaluated in cell-growth inhibition assay with the Molt-3 leukemia cell line in the presence and the absence
of antibody 38C2. The dendritic unit was applied as a platform for a heterodimeric prodrug, which achieved
a remarkable increase in toxicity with its bioactivation.
immolative dendrimers14-17 (SIDs). These unique structural
dendrimers can release all of their tail units through a self-
Introduction
The unique structural properties of dendrimers1,2 increasingly
entice scientists to use them for drug delivery applications.3-7
Recently, biodegradable8,9 and disassembled10,11 dendritic mol-
ecules have been attracting growing attention. Several anticancer
prodrugs have been designed for selective activation in malig-
nant tissues by a specific enzyme, which is targeted12 or secreted
near tumor cells.13 The release of the free drug by a specific
enzyme only takes place upon cleavage of a prodrug protecting
group. The circumstances under which a cleavage event will
release one molecule of free drug may limit the total amount
of the targeted drug, depending on the rate and concentration
of the specific enzyme. We and others have recently reported a
new class of dendritic molecules that were termed self-
immolative chain fragmentation, which is initiated by a single
cleavage event at the dendrimer’s core. Incorporation of drug
molecules as the tail units and an enzyme substrate as the trigger
can generate a multi-prodrug unit that will be activated upon a
single enzymatic cleavage. Self-immolative dendritic prodrugs
may open new opportunities for targeted drug delivery. In
contrast to conventional dendrimers, SIDs are fully degradable
and can be excreted easily from the body. The cleavage effect
of a tumor-associated enzyme or a targeted one could be
amplified and therefore may increase the number of active drug
molecules in targeted tumor tissues.
The AB2 building unit of the dendrimer is based on 2,6-bis-
(hydroxymethyl)-p-cresol (7), a commercially available com-
pound, which has three functional groups (Scheme 1). The two
hydroxybenzyl groups are attached through carbamate linkages
to drug molecules, and the phenol functionality is linked to a
trigger through a short spacer, N,N′-dimethylethylenediamine
(compound 1). The cleavage of the trigger initiates a self-
immolative reaction, starting with spontaneous cyclization of
amine intermediate 2, to form an N,N′-dimethylurea derivative.
The generated phenol 3 undergoes a 1,4-quinone methide
rearrangement, followed by spontaneous decarboxylation to
liberate one of the drug molecules. The quinone methide species
4 is rapidly trapped by a water molecule (from the reaction
† Tel-Aviv University.
‡ Humboldt University.
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J. AM. CHEM. SOC. 2004, 126, 1726-1731
10.1021/ja039052p CCC: $27.50 © 2004 American Chemical Society

Bioactivation of Self-Immolative Dendritic Prodrugs
A R T I C L E S
Scheme 1. Mechanism of Dimeric Prodrug Activation by a Single Enzymatic Cleavage
Scheme 2. Synthesis of the Homodimeric Prodrug of Doxorubicin with a Trigger That Is Activated with Catalytic Antibody 38C2
medium) to form a phenol (compound 5), which again undergoes
a 1,4-quinone methide rearrangement to liberate the second drug
entity. The generated quinone methide species 6 is trapped again
by a water molecule to form 7.
multiple numbers of drug molecules with a single cleavage of
an enzymatic trigger.
Results and Discussion
Homodimeric Prodrugs. The synthesis of the dimeric
prodrugs is described in Schemes 2 and 3. Thus, commercially
available 7 was reacted with 2 equiv of (TBS)Cl to afford phenol
Here we report on the design, synthesis, and bioactivation of
the dendritic prodrug platform, which allows the release of
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A R T I C L E S
Shamis et al.
Scheme 3. Synthesis of the Homodimeric Prodrug of
Camptothecin with a Trigger That Is Activated with Catalytic
Antibody 38C2
Chart 1. Chemical Structure of Prodrugs 14 and 14a
problems of the dimeric prodrug of DOX forced us to use
Cremophor EL as a cosolvent agent. The in vitro assay results
are shown in Figure 1.
8, which was acylated with p-nitrophenyl chloroformate to give
carbonate 9. The latter was reacted with mono-Boc-N,N′-
dimethylethylenediamine to generate compound 10, which was
deprotected in the presence of Amberlyst-15 to give diol 11.
Deprotection with TFA afforded an amine salt, which was
reacted in situ with linker I (activated form of antibody 38C2
substrate; see the Supporting Information for further details) to
generate compound 12. The latter was mixed with 2 equiv of
doxorubicin to afford homodimeric prodrug 14.
Acylation of diol 11 with 2 equiv of p-nitrophenyl chloro-
formate afforded dicarbonate 15, which was then reacted with
2 equiv of camptothecinamine units to give compound 16.
Deprotection with TFA afforded an amine salt, which was
reacted in situ with linker II (see the Supporting Information
for further details) to yield homodimeric prodrug 17.
As the first example of a dimeric prodrug, we chose to
incorporate the anticancer drug doxorubicin,18 and catalytic
antibody 38C219 as the activating enzyme. Antibody 38C2
catalyzes a sequence of retro-aldol retro-Michael cleavage
reactions, using substrates that are not recognized by human
enzymes.20 Therefore, nonspecific prodrug activation should be
minimal. Furthermore, the antibody has demonstrated efficacy
in activating several prodrugs in vitro and in vivo. A dramatic
75% decrease in subcutaneous (sc) tumor size has been observed
in mice that received a combination of intratumoral injections
of antibody 38C2 and systemic treatments with an etoposide
prodrug.21
Solvent controls show that the toxicity of Cremephor EL is
about 50-fold less than that of free DOX and about the same as
those of the prodrugs. Notably, there is still a 50-fold remaining
difference between the IC50 of the prodrugs and free DOX.
When catalytic antibody 38C2 was incubated with the prodrugs,
it was evident that the IC50 of the dimeric prodrug had shifted
closer to the IC50 of the free DOX. Importantly, the toxicities
of the monomeric prodrug and the dimeric prodrug remain in a
similar range, indicating the relative stability for hydrolysis of
the drug molecules’ linkages with the platform.
To avoid the toxicity of Cremephor EL, we synthesized a
camptothecin22 (CPT) dimeric prodrug, which was found to be
much more water soluble than the DOX prodrug 14. The CPT
was attached to the platform through a short self-immolative
linker. The triggering substrate was the same retro-aldol retro-
Michael moiety (Chart 2). The activation of dimeric prodrug
17 was compared with that of a known monomeric CPT
prodrug, 17a, using the Molt-3 cell line growth inhibition assay
(Figure 2).
The IC50 values of the monomeric and the dimeric prodrugs
were found to be almost the same, and the prodrugs were about
200-fold less toxic than free CPT. When catalytic antibody 38C2
was added, both prodrugs were activated. However, while the
activity of the monomeric prodrug had shifted to a 10-fold
difference from that of free CPT, the dimeric prodrug was shown
to be about 4 times more active upon addition of 38C2, meaning
that more toxicity was achieved using the dimeric prodrug and
38C2 in comparison to monomeric prodrug and the same
concentration of antibody.
The retro-aldol retro-Michael substrate of antibody 38C2 was
attached to the adaptor platform through a self-immolative linker
with adequate length, to avoid steric hindrances accompanying
the complex structure of the doxorubicin (DOX) molecule (Chart
1).
The activation of dimeric prodrug 14 was compared with that
of previously reported20 monomeric prodrug 14a, using a cell-
growth inhibition assay of the Molt-3 cell line. Solubility
To the best of our knowledge, these are the first reported
examples of dimeric prodrug bioactivation which involve a
double 1,4-quinone methide rearrangement, to release two
molecules of drug in a single enzymatic event.
Heterodimeric Prodrugs. With these results in hand, we
were motivated to synthesize a heterodimeric prodrug, con-
structed of a combination of DOX and CPT. The synthesis was
performed straightforward as presented in Scheme 4. Thus,
dicarbonate 13 was selectively reacted with 1 equiv of the CPT-
(18) Di Marco, A.; Cassinelli, G.; Arcamone, F. Cancer Treat Rep. 1981, 65
(Suppl. 4), 3-8.
(19) Wagner, J.; Lerner, R. A.; Barbas, C. F., III. Science (Washington, D.C.)
1995, 270, 1797-1800.
(20) Shabat, D.; Rader, C.; List, B.; Lerner, R. A.; Barbas, C. F., III. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 6925-6930.
(21) Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R. A.; Rader, C.; Lerner, R. A.;
Barbas, C. F., III. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7528-7533.
(22) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmar, K. H.; McPhail, A. T.;
Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888-3890.
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Bioactivation of Self-Immolative Dendritic Prodrugs
A R T I C L E S
Figure 1. Growth inhibition assay of the human Molt-3 leukemia cell line, with addition of prodrugs in the presence and absence of catalytic antibody 38C2
(cells were incubated for 72 h): (A) (9) DOX, (b) pro-DOX 14a, (2) pro-DOX 14a + 1 µM 38C2, (1) solvent control; (B) (9) DOX, (b) pro-DOX 14,
(2) pro-DOX 14 + 1 µM 38C2, (1) solvent control.
Figure 2. Growth inhibition assay of the human Molt-3 leukemia cell line, with prodrugs in the presence and absence of catalytic antibody 38C2 (cells were
incubated for 72 h): (A) (9) CPT, (b) pro-CPT 17a, (2) pro-CPT 17a + 1 µM 38C2; (B) (9) CPT, (b) pro-CPT 17, (2) pro-CPT 17 + 1 µM 38C2.
Chart 2. Chemical Structure of Prodrugs 17 and 17a
amine unit to give compound 18. The latter was directly reacted
with 1 equiv of DOX to give the final heterodimeric prodrug
19.
The bioactivation of dimeric prodrug 19 was compared with
that of a 1:1 combination of monomeric prodrugs 14a and 17a
at the appropriate concentrations. The in vitro results using cell-
growth inhibition assay of the Molt-3 cell line are shown in
Figure 3. The IC50 values of both prodrug 19 and the combina-
tion 14a + 17a are almost the same. However, in the presence
of antibody 38C2, the IC50 of prodrugs 14a + 17a shifted to
only 8 nM while the IC50 of prodrug 19 decreased to 0.17 nM.
This effect is remarkable and shows a clear advantage of the
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A R T I C L E S
Shamis et al.
Scheme 4. Synthesis of Heterodimeric Prodrug 19, Containing an Enzymatic Trigger Substrate of Catalytic Antibody 38C2
homodimeric prodrugs (14 and 17) exhibit improved activity
by a factor of 2-4 when compared with their monomeric
counterparts (14a and 17a). This effect can simply be explained
by the number of enzymatic cleavages that are required to
activate the prodrugs. Two cleavage events are needed for two
molecules of monomeric prodrugs, while only one is needed
for a homodimeric unit. This advantage can be achieved only
if undesired toxicity toward normal cells of the dimeric prodrug
will remain in the range of the monomeric one. To our delight,
the chemical stabilities of both monomeric and dimeric prodrugs
in the cell medium without the catalytic antibody were found
to be relatively similar. The mild advantageous effect in
bioactivation of the dimeric prodrug could be increased sig-
nificantly when higher generations of dendritic prodrugs are
generated. We have showed the feasibility of the synthetic
design by preparing a G2-dendron prodrug of CPT with a retro-
aldol retro-Michael substrate for catalytic antibody 38C2.
However, the bioactivation of this dendrimer still requires
additional studies.
Figure 3. Growth inhibition assay of the human Molt-3 leukemia cell line
(cells were incubated for 96 h): (9) pro-CPT 17a + pro-DOX 14a, (b)
pro-CPT 17a + pro-DOX 14a + 1 µM 38C2, (2) heterodimeric prodrug
19, (1) heterodimeric prodrug 19 + 1 µM 38C2.
The best results for the dendritic compounds were obtained
with a heterodimeric prodrug. The toxicity for prodrug 19,
constructed of DOX and CPT, was about 50-fold higher than
activity measured using a combination of two monomeric
prodrugs (14a and 17a) when bioactivation was performed. One
reason for this effect was already discussed before and is merely
related to the number of cleaving events that are required for
activation. However, additional explanation is needed to clarify
the significant effect. At this time, we believe that in the
bioactivation of the two monomeric prodrugs (14a and 17a)
there may be a case of competitive substrates. Thus, if one
prodrug is a better substrate than the other, the overall release
of the free drug combination is inhibited in comparison with
the activation of the heterodimeric prodrug. Importantly, the
toxicity of the heterodimeric prodrug in the absence of catalytic
antibody 38C2 was similar to that measured for the combination
of the monomeric prodrugs.
Table 1. IC50 Values from Cell-Growth Inhibition Assays
a
b
a
b
drug/
IC
IC
drug/
IC
IC
50
50
50
50
prodrug
(nM)
(nM)
prodrug
(nM)
(nM)
DOX
14
14a
CPT
17
6.3
6.3
12.6
26.3
0.13
0.38
17a
28.4
1.5
8.0
143
111
0.13
12.8
14a + 17ac
19c
112
96.5
0.06
0.17
0.06
DOX + CPTc
a Cells were incubated in medium with drug/prodrug. b Cells were
incubated in medium with drug/prodrug + 1 µM catalytic antibody 38C2.
c Cells + prodrugs were incubated for 96 h.
heterodimeric prodrug in comparison to a combination of two
monomeric prodrugs. The prodrug bioactivation is much more
efficient if two drug molecules are attached to a single common
masking enzymatic substrate rather than two separated sub-
strates. Importantly, no drug release was observed when prodrug
19 was incubated in the cells’ extract for 24 h.
The in vitro results of all the cell-growth inhibition assays
are summarized in Table 1.
Conclusions
Bioactivation of homodimeric prodrugs shows a slight but
clear toxicity increase over activation of the monomeric
prodrugs. In the studied examples, both DOX and CPT
In summary, we have demonstrated the first application of
self-immolatve dendrimers as a platform for multi-prodrugs,
activated by a single enzymatic cleavage. We synthesized homo-
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Bioactivation of Self-Immolative Dendritic Prodrugs
A R T I C L E S
and heterodendritic prodrugs of DOX and CPT with a triggering
substrate of catalytic antibody 38C2, which functions as a model
enzyme. The bioactivation of the dendritic prodrugs was
evaluated by growth inhibition assays using the Molt-3 leukemia
cell line, and the prodrugs showed a mild to significant increase
in toxicity in comparison with the classical monomeric prodrugs.
Dimeric units with an enzymatic trigger were readily activated
upon addition of catalytic antibody 38C2, while the tetrameric
counterpart was synthesized but thus far has failed to be a
substrate for the antibody. A remarkable increased effect in
toxicity was observed upon bioactivation of a heterodimeric
prodrug of DOX and CPT. Different drugs can be introduced
on the dendritic platform to achieve synergetic effects, and
precise drug combinations may be tailored for specific types of
cancer. In vivo experiments using this concept will be initiated
after further optimization of the required compounds and
conditions.
Acknowledgment. We thank Dr. Richard A. Lerner and Dr.
Carlos F. Barbas III of The Scripps Research Institute for
providing antibody 38C2. Financial support has been provided
by Tel-Aviv University. Special thanks go to Dr. Michael
Meijler for his helpful comments.
Supporting Information Available: Full experimental details,
characterization data, and cell assay conditions (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA039052P
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