Molecular, Macromolecular, and Supramolecular Glucuronide Prodrugs: Lead Identified for Anticancer Prodrug Monotherapy

Article abstract of DOI:10.1002/anie.201916124

In this work, a tumor growth intervention by localized drug synthesis within the tumor volume, using the enzymatic repertoire of the tumor itself, is presented. Towards the overall success, molecular, macromolecular, and supramolecular glucuronide prodrug

Full text of DOI:10.1002/anie.201916124

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Accepted Article
Title: Molecular, macromolecular, and supramolecular glucuronide
prodrugs: an unexpected lead identified for anticancer prodrug
monotherapy
Authors: Morten Jarlstad Olesen, Raoul Walther, Pier Poier, Frederik
Dagnæs-Hansen, and Alexander N. Zelikin
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201916124
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Molecular, macromolecular, and supramolecular glucuronide
prodrugs: an unexpected lead identified for anticancer prodrug
monotherapy
Morten T. Jarlstad Olesena,b, Raoul Walthera, Pier Paolo Poiera, Frederik Dagnæs-Hansen,c
Alexander N. Zelikina,b*
Abstract: In this work, we perform tumor growth intervention via
localized drug synthesis within the tumor volume, using the enzymatic
repertoire of the tumor itself. Towards the overall success, we design
molecular, macromolecular, and supramolecular glucuronide
prodrugs for a highly potent toxin, monomethyl auristatin E (MMAE).
The lead candidate exhibited a fold difference in toxicity between the
prodrug and the drug of 175, had an engineered mechanism to
enhance the deliverable payload to tumours, and contained a highly
potent toxin such that bioconversion of few prodrug molecules created
concentration of MMAE sufficient for efficient suppression of tumor
growth. Each of these points is highly significant and together afford
the tumor. We focused on the privileged glucuronide scaffold[8]
and engineered molecular, macromolecular, and supramolecular
prodrugs, advancing from the front-line methodologies in the
fields of polymer therapeutics[9] and albumin-based protraction
10]
methodologies[5a,
(Figure 1A,B). All the designed prodrugs
successfully masked the toxicity of the incorporated drug, and all
prodrugs released the drug upon enzymatic bioconversion in vitro,
but only one prodrug provided significant anticancer effect in vivo.
The lead candidate could not be predicted based on existing
literature reports, and could not be identified based on the in vitro
toxicity screens. Results of this study are highly important in
presenting a novel, superior scaffold for prodrug delivery to
tumors. We believe that this scaffold will prove useful to all the
diverse modalities of cancer detection, imaging, and treatment
that rely on enhanced tumor localization of the administered
(pro)drug. [5a, 7]
a
safe, selective anticancer measure, making tumor-targeted
glucuronides attractive for translational medicine.
Systemic toxicity is the single most important determinant of
success or failure of anticancer drugs. Side effects due to the
systemic drug exposure exert the upper limit of the achievable
plasma drug concentration, often well below the optimized in vitro
dose. A powerful approach to overcome this is via a localized
activation of prodrugs at the site of action. [1] Such localized drug
synthesis has been accomplished via the “enzyme prodrug
therapies” (EPT) differed by the approach to localize the enzyme
at the tumor site. Most notably, this has been accomplished using
antibodies, [2] localized gene expression, [3] and/or via engineering
enzymes into implantable biomaterials [4]. An elegant, unique
strategy for therapeutic cancer intervention lies in using the
enzymatic repertoire of the tumor itself, via an innate, disease-
mediated EPT (i-EPT, Scheme 1). [5] The tumor microenvironment
is rich in extracellular enzymes that in healthy tissues are confined
to the intracellular compartments. [6] This phenomenon provides a
high specificity of enzyme localization in the body, for a highly
localized prodrug activation. [7]
Delivery of an enhanced injected dose to tumors can rely on
the tools of active and/or passive targeting. Active, receptor-
mediated targeting can be achieved using antibodies and small
molecule conjugates, of which the former appear to be
advantageous.[11] However, active targeting is suited only to the
well-characterized cancers with a documented phenotype. In turn,
passive targeting is advantageous in that it relies on non-specific,
broadly applicable methodologies of decreasing renal excretion of
the injected dose, and/or the tumor-associated “enhanced
permeation and retention” (EPR) effect, which together facilitate
accumulation of the injected dose within the tumor.[12]
[9a]
Macromolecular
nanomedicine
and supramolecular tools of
[13]
are academically successful in their own right
and present a wide landscape of opportunities for drug delivery
via EPR. Of these, PEGylation is arguably the most successful
synthetic methodology to produce macromolecular (pro)drugs
Herein, we reveal that key to a successful i-EPT (cancer
prodrug monotherapy) lies in maximizing the prodrug delivery to
[a]
[b]
[c]
Dr. M.T. Jarlstad Olesen, Dr. R. Walther, P.P. Poier, Dr. A.N. Zelikin
Department of Chemistry, Aarhus University, Aarhus, Denmark
E-mail: zelikin@chem.au.dk
Dr. M.T. Jarlstad Olesen, Dr. A.N. Zelikin
iNano Interdisciplinary Nanosciece Centre,
Aarhus University, Aarhus, Denmark
F. Dagnæs-Hansen
Department of Biomedicine, Aarhus University, Aarhus, Denmark
Supporting information for this article is given via a link at the end of
the document.
Scheme 1. Schematic illustration of the innate Enzyme Prodrug therapy (i-
EPT).
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Figure 1. Prodrugs structures, synthesis scheme, and bioconversion. (A) Schematic illustration of the key synthetic steps in the synthesis of the prodrugs of
MMAE employed in this work, and the mechanism of triggered release; For detailed synthetic procedures and compound characterization details, see
supplementary materials; (B) Chemical structures of the protraction arm R in the structure of the prodrugs of MMAE; (C) normalized HPLC chromatograms for
prodrug 4 (20 mg/L) illustrating prodrug stability upon incubation in phosphate buffered saline for a period of at least 24 h and drug recovery within 2 h of
incubation in the presence of β-glucuronidase (1 mg/L) at 37ºC; (D) Quantitative evaluation of the stability and hydrolysis of prodrug 4 in the presence or absence
of β-glucuronidase from C, data represented as mean ± SD of 3 independent experiments (N=3)
with optimized pharmacokinetics, for both biological [14] and small
auristatin E (MMAE), one of the most potent toxins used in
[15]
[22]
molecule
therapeutics. PEGylation serves to increase the
medicinal practice.
Finally, the protraction arm R was
hydrodynamic radius of the molecule, which leads to impede renal
introduced at a late stage of the synthesis and varied between
PEG 2,000 Da and PEG 20,000 Da (tools of polymer
filtration, to limit non-specific tissue penetration, and to facilitate
[16]
accumulation in tumors via the EPR effect.
An equally
therapeutics),
as
well
as
1,2-distearoyl-sn-glycero-3-
successful biological counterpart is albumin, the most abundant
protein in human plasma, characterized with a phenomenal
phosphoethanolamine (DSPE), 4-(p-iodophenyl)butyric acid
(AlbuTag), and trityl group (Tr), of which the latter three are known
albumin binders. [10b, 23]
Synthesis of prodrugs started from the commercially
available 1,2,3,4-tetra-O-acetyl-β-D-glucuronide methyl ester 1
circulation half-life of around three weeks. [17] Drug conjugates [18]
[10b]
and supramolecular non-covalent adducts
with albumin are
successful in their own right, of which the latter are increasingly
popular due to decreased handling of the biomolecule and the
incredibly broad spectrum of synthetic opportunities to define
affinity to albumin and the pharmacokinetics of the therapeutic
molecule. [10a] Indeed, the overall majority of covalent conjugates
to albumin pursue the same chemistry of maleimide-to-Cys34
conjugation.[19] In contrast, the arsenal of non-covalent binders
contains dozens of ligands and spacers with associated
(Figure 1A and SI1). First step included the formation of the
[24]
extended scaffold
via chemical O-glycosylation of 4-hydroxy-
3-nitrobenzaldehyde (2).[6c] Subsequently, the aldehyde
functionality and the nitro group were reduced stepwise to the
benzylic alcohol and an aniline (3), respectively. Acylation of
aniline was then performed to introduce the alkyne or a primary
amine functionality. Conjugation of the MMAE toxin was achieved
with the use of 4-nitrophenyl chloroformate chemistry.[25] Finally,
conjugation of the protraction arm R was developed as the late
stage diversification, performed via either a direct reaction with
activated esters or the copper catalysed azide-alkyne
cycloaddition.
[10b,
opportunities to fine-tune pharmacokinetics of the (pro)drug.
20]
We engineered a library of molecular, macromolecular, and
supramolecular prodrugs using
a modular, self-immolative
prodrug scaffold based on o-substituted p-hydroxybenzyl alcohol
(PHBA) (Figure 1A). [21] This scaffold is unique in that it presents
three sites of modification to install i) the effector molecule, ii) the
trigger for drug release, and iii) the protraction arm.[5a] We focused
Using this general methodology, we obtained a total of eight
prodrugs (4-11) with envisioned diversity of pharmacokinetic
properties (Figure 1B). Molecular prodrugs (4-6) are simplest by
on the glucuronide prodrugs due to their privileged position in EPT. structure, are highly water soluble, and are expected to have
[8]
[26]
Glucuronides are well water-soluble and typically have
shortest blood residence time of the synthesized prodrugs.
restricted cell entry. The latter attribute endows glucuronides with
the highest documented change in toxicity between the prodrug
and the drug (i.e. QIC50, calculated as a fold-ratio between the
Macromolecular prodrugs (7-8) are engineered via PEGylation, a
strategy that limits renal excretion of the molecule by virtue of
having an increased hydrodynamic radius and also enhanced
tumor accumulation via the EPR effect. [16] Finally, supramolecular
[8]
corresponding IC50 values). As a drug, we used monomethyl
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which PEG presents a sufficient spacer between albumin and the
glucuronic acid.
Table 1. Toxicity related IC50 data for the glucuronide prodrugs against
triple-negative human MDA-MB-231 breast adenocarcinoma cells in the
absence or presence of β-glururonidase (- Enz / + Enz, respectively).
QIC50 denotes the toxicity ratio between non-activated and enzyme
activated prodrug: QIC50 = IC50 (- Enz) / IC50 (+ Enz). Toxicity of MMAE in
these experiments was 1.1±0.5 nM.
For successful i-EPT, the envisioned mechanism of prodrug
bioconversion relies on the endogenous, rather than the
externally administered enzyme. We verified if the enzymatic
repertoire of the cancer cells is sufficient for prodrug
bioconversion. Cancer xenografts based on triple-negative MDA-
MB-231 breast adenocarcinoma cells were explanted from mice
and incubated in PBS in the presence of resorufin-β-D-
glucuronide (ResoGlcA), a fluorogenic probe employed such that
increase in fluorescence intensity indicates enzymatic
bioconversion of the probe. Explanted xenografts became well
fluorescent when cultured in the presence ResoGlcA, providing
an ex vivo validation of i-EPT (imaging modality) (Figures
S7,10,11). This was also true in the case of xenografts based on
non-invasive human breast adenocarcinoma MCF-7 cells (ER+,
PR+, HER2-, luminal subtype A) [27] and these too afforded reliable
conversion of the fluorogenic glucuronide (Figures S7,10,11).
Thus, in vivo grown cancerous tissue is capable of converting
glucuronide prodrugs, which is the most important pre-requisite
for the success of i-EPT.
Prodrug R=
Alkyne (4)
H (5)
IC50 (- Enz)
IC50 (+ Enz)
QIC50
255
260
9.4
0.6
2.2
5.4
1.5
2.4
3.3
67.4
425
118
1,7
7
77
175
47
PEG3k (7)
PEG20k (8)
DSPE-PEG (9)
Tr-PEG (10)
AlbuTag (11)
10.5
184
579
1746
prodrugs (9-11) are designed such as to benefit from the
beneficial pharmacokinetic characteristics of albumin, including
extended blood residence time and potential increased tumor
accumulation. [18]
HPLC release studies revealed that all synthesized
glucuronide prodrugs exhibit high stability in physiological buffer
(phosphate buffered saline) for at least 24 h at 37ºC, a feature
that is highly important to minimize the non-specific drug release
(Figure 1C and D and supplementary materials for the remaining
prodrugs). All prodrugs released MMAE quantitatively in presence
of β-glucuronidase, revealing that protraction arms R do not
hinder enzymatic prodrug activation.
Cell culture evaluation of the prodrugs was conducted using
highly metastatic triple-negative (oestrogen receptor (ER)-,
progesterone receptor (PR)-, human epidermal growth factor
receptor 2 (HER2) negative; claudin-low subtype) MDA-MB-231
human breast adenocarcinoma cells
responsiveness (Figure S7 and Table 1). For each prodrug,
toxicity was monitored in the presence or absence of the
activating enzyme, allowing 72 h for the toxin to exert its effect.
Under these conditions, over multiple independent runs, pristine
MMAE revealed an IC50 value of 1.1±0.5 nM. In agreement with
their design, the extended scaffold glucuronides were significantly
less toxic than MMAE whereas toxicity was restored via
enzymatic bioconversion and ensuing drug release, with QIC50
values as high as 425. Interestingly, prodrug structure and
specifically the protraction arm R had a profound effect on the IC50
of the prodrugs, their ability to undergo enzymatic bioconversion,
and the resulting QIC50 values. The first observation is that
PEGylated prodrugs (7, 8), surprisingly, were the least effective
in masking toxicity of MMAE and exhibited IC50 values around 10
nM. Corresponding values for all other prodrugs, including the
albumin-affine DSPE-PEG (9) and Tr-PEG (10), were well over
In vivo anticancer effects mediated by the prodrugs were
quantified in the subcutaneous (s.c.) MDA-MB-231 ectopic
xenografts. We employed a once-weekly s.c. administration of the
prodrugs in line with the growing understanding that this route of
administration considerably decreases treatment costs while
providing patient convenience (home vs hospital administration).
[28] Pristine MMAE, in agreement with prior reports on the subject,
[5a]
proved to be highly toxic and exhibited no curative effects
[27]
with limited treatment
(Figure 2 A1,B1). Molecular prodrug (R=H, 5) significantly masked
systemic toxicity of MMAE and was well tolerated, as evidenced
by the survival rate and the body weight of mice, (Figure 2 A1, B1)
but afforded no discernible anticancer effect (Figure 2 C1, D1).
Same holds true for the majority of macromolecular (PEG3k (7))
and supramolecular (DSPE (9), AlbuTag (11)) prodrugs tested in
this work, and while toxicity of treatment was much improved over
MMAE monotherapy, improvement in the therapeutic effect was
not observed (Figure 2C1,2,D1,2). This observation was rather
surprising and indicates that otherwise successful tools of
[23a]
[10b]
nanomedicine, such as DSPE-PEG,
PEG, and AlbuTag
failed to facilitate translocation of the glucuronide prodrugs to the
tumor in sufficient quantities required for a successful i-EPT, at
least with the chosen sparse drug administration schedule.
Worthy of note, the prodrug with a PEG20k (8) protraction arm
exhibited statistically significant suppression of the tumor growth
rate and this prodrug scaffold deserves further optimization.
Our study reveals an unexpected lead formulation that
afforded statistically significant suppression of tumor growth,
namely the Tr-PEG glucuronide prodrug 10 (Figure 3C1,2,D1,2).
The prodrug based on Tr-PEG (10) was highly active in vivo while
its close analogue without an albumin binding group (R = PEG3k
(7)) was not, providing evidence that albumin binding is key to the
observed anticancer effect. However, prodrugs based on DSPE-
PEG (9) and AlbuTag (11), both albumin affine, were ineffective,
100 nM. PEGylation is
a validated method to minimize
translocation of drugs across biological membranes (e.g.
Naloxegol is a PEGylated, peripherally active opioid).[15] Apparent
increase in toxicity for PEGylated glucuronides 7, 8 (compared to
the simplified, parent prodrugs alkyne (4), R=H (5)) is therefore
surprising. From a different perspective, our data demonstrate
that all the prodrugs underwent efficient bioconversion, which
resulted in the restored cytotoxicity of MMAE. However, the
AlbuTag-functionalized glucuronide stands out as the prod rug
with impeded bioconversion, as evidenced by a relatively high
IC50 = 67 nM observed for the prodrug in the presence of β-
glucuronidase. A likely explanation to this is that AlbuTag is a
relatively short spacer, in which case albumin creates a steric
shield and hinders enzymatic activity on the prodrug. This effect
was not observed for DSPE-PEG (9) or Tr-PEG (10) prodrugs for
signifying that albumin affinity as such is not sufficient for success.
[10, 23a]
Our results, and those from others,
suggest that albumin
binders are not the same with regards to the in vivo performance
of the resulting supramolecular adducts, be it for delivery of
vaccines or anticancer drugs. Most importantly, it appears that in
vitro toxicity screens fail to provide predictive knowledge and
nominate a priori leads for in vivo evaluation. Specifically, in our
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Figure 2. In vivo analysis of toxicity and anticancer activity revealed by the prodrugs of MMAE in ectopic human triple-negative MDA-MB-231 adenocarcinoma
xenografts: (A1,2) mice body weight; (B1,2) Kepler-Meyer survival plots; (C1,2) tumor growth data; (D1,2) end-point statistical evaluation of the tumor growth at day
41 (D1) or 36 (D2). BALB/c-nu mice (n=8 (top panel) or n=6 (lower panel) per group) were inoculated s.c. with 3×106 MDA-MB-231 cells dorsally on the lower
right flank. Arrows in panels C indicate drug administration (s.c., 1.5 mg/kg MMAE equivalents). Statistical significance was evaluated using a mixed-effects
analysis (REML) followed by a Dunnett multiple comparisons test (C1,D1) or a two-way ANOVA followed by Tukey’s multiple comparisons test (C2,D2). P > 0.05
(ns), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****). For panel C1,2 the asterisks indicate statistical comparison between Tr-PEG treated mice and
buffer-dosed mice
hands, prodrugs that performed best in vitro (e.g. exhibited the
highest QIC50 or lowest toxicity of the non-activated prodrug) were
not featured as leads after in vivo evaluation.
based on Tr-PEG, which in turn was very similar to the DSPE-
PEG counterpart. In other words, these pharmacokinetic
parameters appear to hold no predictive value with regards to the
anticancer effectiveness of the corresponding prodrugs of MMAE.
However, we also observed that while the probe based on PEG3k
had the highest blood residence time, it was the counterpart
based on Tr-PEG that exhibited the highest tumor accumulation,
followed by probes based on PEG3k and DSPE-PEG (Figure 3B).
In our hands, tumor accumulation is the only pharmacokinetic
parameter that shows correlation with the in vivo anticancer
effects.
Next, we analyzed pharmacokinetics of selected prodrugs
(based on PEG3k, Tr-PEG, and DSPE-PEG). For this, imaging
reagents were designed to contain the Cy7 fluorophore instead of
the MMAE toxin. Imaging reagents were administered s.c.,
following which fluorescence was quantified in periodically
collected blood samples. Characteristic blood content profiles
(Figure 3A) were used to calculate the pharmacokinetics
parameters (Table 2), specifically Tmax (time to maximum plasma
concentration), Cmax (plasma concentration at maximu m), AUC
Analysis of the structure-activity relationship for the panel of
molecular, macromolecular, and supramolecular prodrugs
employed in this work reveals the Tr-PEG protraction arm to be
the most effective tool to achieve enhanced tumor localization of
the prodrug with ensuing tumor growth suppression. The likely
mechanism of this pharmacokinetic phenomenon is that this
prodrug associates non-covalently with albumin, as is the case
for the DSPE and AlbuTag counterpart.[10b, 28] However, affinity
of trityl group to albumin to this point a conjecture as only limited
prior art exists to support this notion.[29] In an effort to validate
such affinity, we performed in silico molecular docking
simulations for albumin binding with trityl thioether as well as a
panel of other known albumin binders, and considered two major
ligand binding sites for albumin (the so-called Sudlow I and II
sites). Computer modelling revealed no docking for trityl
thioether to Sudlow II site. In stark contrast, docking to Sudlow I
site was successful, with a docking score similar to a known
Sudlow I binder indomethacin (for numerical values, see Tables
S5 and S6). Docking simulations suggest that trityl ether binds
such that it essentially superimposes with the most well-
characterized Sudlow I ligands (which also give the lowest
docking score, that is, have highest computed affinity), namely
(area under curve), T β (plasma elimination half-life during the
½
elimination phase), and Kel (elimination rate constant). Analysis of
these data reveals that the imaging probe based on PEG3k
exhibited the highest Tmax, Cmax, and AUC, followed by the prodrug
Table 2. Pharmacokinetic parameters for the imaging probes containing PEG3k
± Tr or DSPE-PEG protraction arm and Cy7 as an imaging fluorophore (n=4).
BALB/cJRj mice were injected in the scruff with 0.4 mg/kg Cy7 labelled DSPE
or PEG3k ± Tr in a fluorescently matched dose. Buffer containing 4.8% DMSO
was administered as control. Cy7 signal was analyzed over time from the
plasma fraction of blood samples taken from the tail at the indicated time points.
Data
refers to
AUC5min-96h Cmax
Tmax Kel
(h)
agent
T1/2 β (h)
24.2
(a.u.)
(a.u.)
(h-1)
Fig. 3A
DSPE-Cy7
PEG-Cy7
Tr-PEG-Cy7
PEG-Cy7
0.9
4.61
2
0.029
0.044
0.047
0.046
Fig. 3A
2.3
8.82
3.78
4.95
5
2
5
15.9
Fig. S12B
Fig. S12B
0.58
1.25
14.7
15.0
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Figure 3. Pharmacokinetics of prodrugs. (A) Representative
pharmacokinetics profiles for the imaging probes containing PEG3k or DSPE-
PEG protraction arm and Cy7 as an imaging fluorophore. (BALB/cJRj mice, s.c.
dose administration. For detail, see Supporting Information. The results are
expressed as means ± SD, N=4) (B) Quantitative tumor localization data
obtained in two consecutive experiments for pairwise comparison of PEG3k with
DSPE-PEG and PEG3k and Tr-PEG (BALB/c-nu mice, MDA-MB-231 xenografts
s.c. dose administration. For detail, see Supporting Information. The results are
represented as means of quadruplets ± SD (n = 4). Statistical significance was
evaluated using an unpaired t-test. P ≤ 0.05 (*), P ≤ 0.01 (**).
Figure 4. Computational modelling of binding between albumin and ligands.
Structure of albumin co-crystallized with phenylbutazone (orange) (PDB code:
2BXC) binding to Sudlow’s site I (left side). Superpositions of phenylbutazone
(orange), (R)-warfarin (teal) and methyl trityl thioether (green) are presented
in their putative binding mode to Sudlow’s site I and displayed from three
different angles (right side). For SP values (docking scores), see Table S5
(Sudlow’s site I) and Table S6 (Sudlow’s site II).
phenylbutazone and warfarin, Figure 4. Thus, computer
simulations strongly suggest that trityl ether is a good albumin
binder, likely endowing the conjugated cargo with albumin
hitchhiking property in vivo.
anticancer drugs, MMAE is phenomenally potent such that
bioconversion of few prodrug molecules creates the concentration
of MMAE sufficient for efficient cell killing - and with a pronounced
by-stander effect to affect the tumor volume. Each of these points
is significant and together, these attributes render tumor targeted
glucuronides attractive for translational studies. We are now
optimizing drug dose and administration frequency to optimize the
anticancer effects.
Taken together, this study is important in that we designed
and compared side-by-side molecular, macromolecular, and
supramolecular glucuronide prodrugs for MMAE to achieve
localized, autonomous bioconversion within the tumor volume
with ensuing cancer growth suppression. All prodrugs significantly
masked toxicity of MMAE, as observed both in vitro and in in vivo,
and all prodrugs released MMAE upon bioactivation. Remarkably,
significant variations were observed with regards to IC50 and
QIC50 for the prodrugs in vitro, and even greater difference was
observed in vivo, in which case only two prodrugs exhibited
statistically significant cancer growth suppression and one
formulation stood out as a lead. The identified lead could not be
predicted from the in vitro toxicity screens or in vivo derived
Acknowledgements. ANZ acknowledges funding from the
European Research Council Consolidator Grant (ERC-2013-CoG
617336 BTVI); PPP acknowledges Danish Council for
Independent Research (Grant No. 4181-00030).
References
[1] B. Städler, A. N. Zelikin, Adv. Drug Deliv. Rev. 2017, 118, 1.
[2] S. K. Sharma, K. D. Bagshawe, Adv. Drug Deliv. Rev. 2017,
118, 2-7.
[3] R. Mooney, A. Abdul Majid, J. Batalla, A. J. Annala, K. S.
Aboody, Adv. Drug Deliv. Rev.2017, 118, 35-51.
pharmacokinetics parameters (total drug exposure AUC, Cmax
,
Tmax and plasma half-life T1/2 β). When comparing imaging probes
and anticancer prodrugs with the same protraction arm, we find
that anticancer effects correlate only with one other parameter,
namely extent of the tumor localization of the probe.
[4] a) B. Fejerskov, M. T. Jarlstad Olesen, A. N. Zelikin, Adv.
Drug Deliv. Rev. 2017, 118, 24-34; b) M. T. J. Olesen, A. K.
Winther, B. Fejerskov, F. Dagnaes-Hansen, U. Simonsen, A. N.
Zelikin, Adv. Therap. 2018, 1, 1800023.
[5] a) Renoux, F. Raes, T. Legigan, E. Peraudeau, B. Eddhif,
P. Poinot, I. Tranoy-Opalinski, J. Alsarraf, O. Koniev, S. Kolodych,
S. Lerondel, A. Le Pape, J. Clarhaut, S. Papot, Chem. Sci. 2017,
8, 3427-3433; b) S. Cazzamalli, B. Ziffels, F. Widmayer, P. Murer,
G. Pellegrini, F. Pretto, S. Wulhfard, D. Neri, Clin. Cancer Res.
2018, 24, 3656-3667.
[6] a) B. Sperker, J. T. Backman, H. K. Kroemer, Clin. Pharmac.
1997, 33, 18-31; b) D. Weyel, H. H. Sedlacek, R. Müller, S.
Brüsselbach, Gene Ther. 2000, 7, 224; c) K. Bosslet, R. Straub,
M. Blumrich, J. Czech, M. Gerken, B. Sperker, H. K. Kroemer, J.-
P. Gesson, M. Koch, C. Monneret, Cancer Res. 1998, 58, 1195-
1201.
From a translational perspective, we believe that the
designed prodrugs offer several decisive points of advantage.
First, glucuronide conversion within the tumor volume has
recently been shown to be a highly specific process, so much so
that it can be used for cancer detection7, illustrating inherent
safety of cancer prodrug monotherapy with the use of
glucuronides. In turn, the QIC50 values we record for glucuronides
are significantly higher than those for e.g. hypoxia activated
prodrugs (HAP) that were evaluated and failed in multiple clinical
trials [30] (QIC50 under 10 for HAP, over 175 for the lead candidate
engineered in this work). In other words, our prodrugs are
significantly more selective and safer. Furthermore, prodrugs
developed in this work have an engineered mechanism to
enhance the deliverable payload to tumors, further contributing to
the safety of this platform. Finally, in stark contrast with
micromolar potency of HAP, doxorubicin and many other
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[7] a) A. N. Zelikin, Nat. Chem. 2019, doi: 10.1038/s41557-019-
0400-0; b) J. Lange, B. Eddhif, M. Tarighi, T. Garandeau, E.
Péraudeau, J. Clarhaut, B. Renoux, S. Papot, P. Poinot, Angew.
Chem. Int. Ed. 2019, 58, 17563-17566.
[8] R. Walther, J. Rautio, A. N. Zelikin, Adv. Drug Deliv. Rev.
2017, 118, 65-77.
[9] a) I. Ekladious, Y. L. Colson, M. W. Grinstaff, Nat. Rev. Drug
Discov. 2019, 18, 273-294: b) C. F. Riber, A. N. Zelikin, Curr. Op.
Coll. Interf. Sci. 2017, 31, 1-9.
[10] a) J. Lau, P. Bloch, L. Schaffer, I. Pettersson, J. Spetzler, J.
Kofoed, K. Madsen, L. B. Knudsen, J. McGuire, D. B.
Steensgaard, H. M. Strauss, D. X. Gram, S. M. Knudsen, F. S.
Nielsen, P. Thygesen, S. Reedtz-Runge, T. Kruse, J. Med. Chem.
2015, 58, 7370-7380; b) J. Lau, O. Jacobson, G. Niu, K.-S. Lin, F.
Bénard, X. Chen, Bioconj. Chem. 2019, 30, 487-502.
[11] S. Cazzamalli, A. Dal Corso, F. Widmayer, D. Neri, J. Am.
Chem. Soc. 2018, 140, 1617-1621.
[12] S. K. Golombek, J. N. May, B. Theek, L. Appold, N. Drude,
F. Kiessling, T. Lammers, Adv. Drug Deliv. Rev. 2018, 130, 17-
38.
[13] J. Nam, S. Son, K. S. Park, W. Zou, L. D. Shea, J. J. Moon,
Nat. Rev. Mater. 2019.
[14] A. N. Zelikin, C. Ehrhardt, A. M. Healy, Nat. Chem. 2016, 8,
997.
[15] K. P. Garnock-Jones, Drugs 2015, 75, 419-425.
[16] R. B. Greenwald, Y. H. Choe, J. McGuire, C. D. Conover,
Adv. Drug Deliv. Rev 2003, 55, 217-250.
[17] J. T. Sockolosky, F. C. Szoka, Adv. Drug Deliv. Rev. 2015,
91, 109-124.
[18] L. Pes, S. D. Koester, J. P. Magnusson, S. Chercheja, F.
Medda, K. Abu Ajaj, D. Rognan, S. Daum, F. I. Nollmann, J.
Garcia Fernandez, P. Perez Galan, H. K. Walter, A. Warnecke, F.
Kratz, J. Controlled Release 2019, 296, 81-92.
[19] B. Elsadek, F. Kratz, J. Controlled Release 2012, 157, 4-28.
[20] C. Müller, R. Farkas, F. Borgna, R. M. Schmid, M. Benešová,
R. Schibli, Bioconj. Chem 2017, 28, 2372-2383.
[21] P. L. Carl, P. K. Chakravarty, J. A. Katzenellenbogen, J.
Med. Chem. 1981, 24, 479-480.
[22] P. D. Senter, E. L. Sievers, Nat. Biotechnol. 2012, 30, 631.
[23] a) H. Liu, K. D. Moynihan, Y. Zheng, G. L. Szeto, A. V. Li, B.
Huang, D. S. Van Egeren, C. Park, D. J. Irvine, Nature 2014, 507,
519-522; b) W. Liu, J. Nie, X. Tan, H. Liu, N. Yu, G. Han, Y. Zhu,
F. A. Villamena, Y. Song, J. L. Zweier, Y. Liu, The Journal of
Organic Chemistry 2017, 82, 588-596; c) Y. Song, Y. Liu, C.
Hemann, F. A. Villamena, J. L. Zweier, J. Org. Chem. 2013, 78,
1371-1376.
[24] R. Walther, M. T. Jarlstad Olesen, A. N. Zelikin, Org. Biomol.
Chem. 2019, 17, 6970-6974.
[25] B. Sammet, Synlett 2009, 2009, 3050-3051.
[26] A. V. Stachulski, X. Meng, Nat Prod. Reports 2013, 30, 806-
848.
[27] Y. L. Liu, C. K. Chou, M. Kim, R. Vasisht, Y. A. Kuo, P. Ang,
C. Liu, E. P. Perillo, Y. A. Chen, K. Blocher, H. Horng, Y. I. Chen,
D. T. Nguyen, T. E. Yankeelov, M. C. Hung, A. K. Dunn, H. C.
Yeh, Sci. Rep. 2019, 9.
[28] A. C. Anselmo, Y. Gokarn, S. Mitragotri, Nat. Rev. Drug
Discov. 2018, 18, 19.
[29] Y. Song, Y. Liu, W. Liu, F. A. Villamena, J. L. Zweier, RSC
Adv. 2014, 4, 47649-47656.
[30] W. R. Wilson, M. P. Hay, Nat. Rev. Cancer 2011, 11, 393-
410.
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Entry for the Table of Contents
Safe, tumor-accumulating prodrugs that
are specific to the enzymatic repertoire
of the tumor comprise an effective
anticancer therapy. The lead structure
could not be predicted by the in vitro / ex
vivo screens but was identified through in
vivo monitoring
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