Reversing the undesirable pH-profile of doxorubicin: Via activation of a di-substituted maleamic acid prodrug at tumor acidity

Article abstract of DOI:10.1039/c7cc06843c

The acid-labile behavior of di-substituted maleamic acid (DMA) and its equilibrium with di-substituted maleimide (DMI) are exploited to build an ultra acid-sensitive, small molecule prodrug that can be activated by tumor extracellular pH (pHe) in the range of 6.5-6.9. Such a DMA prodrug reversed the unfavorable pH-profile of doxorubicin (Dox), which may improve its therapeutic window.

Full text of DOI:10.1039/c7cc06843c

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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Reversing the undesirable pH-profile of doxorubicin via activation
of di-substituted maleamic acid prodrug at tumor acidity
Anqi Zhang,a Lan Yaob and Ming An*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
tumor pHe.29ꢀ34 Besides DMA the most acidꢀlabile linker known is
The acid-labile behavior of di-substituted maleamic acid (DMA)
and its equilibrium with di-substituted maleimide (DMI) are
exploited to build an ultra acid-sensitive, small molecule prodrug
that can be activated by tumor extracellular pH (pHe) in the range
of 6.5-6.9. Such DMA prodrug reversed the unfavorable pH-profile
of doxorubicin (Dox), which may improve its therapeutic window.
35
the spiro diorthoester,30,
which would involve more elaborate
chemistry for drug conjugation and traceless release. For amine
drugs such as Dox, since DMA is an amide, it constitutes a natural
traceless linker with one step conjugation chemistry.
Dox is a prominent chemotherapy drug (chemoꢀdrug) commonly
used in the treatments of breast and ovarian cancers, childhood solid
tumors, sarcomas and other malignancies.36, 37 Unfortunately, Dox
and related anthracyclines also cause cumulative, irreversible
myocardial damage that is often fatal.38ꢀ42 The occurrence of Doxꢀ
induced congestive heart failure increases dramatically over only
twoꢀfold difference in accumulated dose, from 1ꢀ2% at 300 mg / m2
body surface area (BSA) to 20ꢀ30% at 600 mg / m2 BSA.38, 40, 42 Dox
intercalates into DNA and inhibits topoisomerase II, which lead to
cell death by a variety of mechanisms.36, 43, 44 To kill cancer cells
Dox molecules must cross cell membrane(s) and accumulate in
nucleus. The amino group of Dox is protonated with a pKa of 8.2.45
Low tumor pHe can impede the cellular uptake of Dox by increasing
the proportion of the drug in the protonated ammonium form, which
is positively charged and less membrane permeable.46ꢀ48 Thus, Dox
is more cytotoxic at pHe of 7.4 than 6.5. Such undesirable pHꢀprofile
is observed at concentrations and time scales relevant to Dox
pharmacokinetics (PK),49 which may reduce drug efficacy and
exacerbate side effects. Many other amine chemoꢀdrugs (e.g. other
anthracyclines, topotecan, vinka alkaloids) also show similar poor
pHꢀprofile.13, 14, 23, 48, 50, 51 The DMA prodrug approach aims to offset
the undesirable pHꢀprofile by selective drug release outside of
cancer cells. A polar modulator (e.g. glutathione in this study) is
attached to Dox through the DMA linker to make the prodrug
membrane impermeable, and thus, inactive.
The DMA group occupies a privileged position among all acidꢀlabile
linkers because it can cleave in response to slight acidity at pH 6.5.1ꢀ7
As such DMA linker may find wide applications in situations where
an ultraꢀresponsive pHꢀsensor is needed in the form of a cleavable
bond. Previously, DMA linker has only been used in the context of
macromolecules (polymers, proteins),2, 3, 7 mainly for pHꢀtriggered
8ꢀ12
charge reversal for nanoparticle engineering.4ꢀ6,
Due to the
macromolecular nature of these DMAꢀpolymer/protein conjugates,
chemical details of DMA formation, conjugate stability and pHꢀ
dependent cleavage (e.g. yields, sideꢀproducts) were limited. In this
study, DMA chemistry is examined in detail to build a targeted small
molecule prodrug that can be selectively activated by tumor acidity.
Fast growth and altered metabolism of cancer cells lead to local,
interstitial acidosis in many tumors, with pHe in the range of 6.5ꢀ
7.0.13ꢀ18 In comparison, pHe of healthy tissues and the intracellular
18, 19
pH of cancer cells usually fall in the range of 7.2ꢀ7.6.13ꢀ16,
Acidosis is more prominent in aggressive tumors and facilitates drug
resistance and metastasis.20ꢀ23 Selective drug release in response to
tumor acidosis may increase the therapeutic window by reducing
exposure of healthy cells to drugs.24 Previous efforts in tumor pHe
triggered drug delivery have focused on complex polymer
24ꢀ27
nanoparticles.8ꢀ12,
In contrast we aim to develop pHꢀsensitive
small molecule prodrugs, which may have the following advantages
over nanoparticleꢀbased systems: (a) Their simple, chemically
defined structures can more easily meet Good Manufacturing
The synthesis of DMA prodrug 8 is outlined in Scheme 1. Acid 1
and acyl chloride 2 were prepared following the methods of Isobe.52
Acyl chloride 2 reacted with Sꢀ(2ꢀpyridylthio)ꢀcysteamine 3 to give
the crosslinker 4 in 50% yield from acid 1. This new compound 4
has general utility for crosslinking an amine group with a thiolꢀ
bearing partner through DMA. Combining 4 with Dox (HCl salt) in
the presence of 4ꢀdimethylaminopyridine (DMAP) and pyridine
quickly led to the formation of DMA regioisomers 5a/b in minutes,
which slowly converted to the ringꢀclosed DMI 6 over a period of
Practice standards, which is
a
significant difficulty28 in
nanomedicine (e.g. 2011ꢀ14 shortage of Doxil, i.e. PEGylated
liposomal Dox, in USA); (b) the extracellular drug release
mechanism of the prodrug is straightforward, while endosomal
trapping of drug payload is a major concern for nanoparticle drug
carriers;28 (c) the pHꢀresponse may be more easily tuned via
structureꢀactivity relationship studies in small prodrugs.
A critical challenge in tumor acidosis activated drug release is the
small difference in pHe between tumor and healthy tissues. Other
acidꢀsensitive linkers, such as ketal, acetal, hemianimal (e.g. Nꢀ
ethoxybenzylimidazole), imine, hydrazone, and monoꢀsubstituted
maleamic acid (e.g. cisꢀaconityl) may respond to lysosomal acidity
of pH 4.5ꢀ6.0, but they are not sensitive enough for triggering by
a.Department of Chemistry, State University of New York (SUNY), Binghamton
University, P.O.Box 6000, Binghamton, New York 13902, USA. E-mail:
This journal is © The Royal Society of Chemistry 20xx
aming@binghamton.edu; Tel: +1 (607) 777 3224
J. Name., 2013, 00, 1-3 | 1
b.Department of Physics, SUNY, Binghamton University, P.O.Box 6000, Binghamton,
New York 13902, USA.
† Electronic Supplementary Information (ESI) available: Tables S1-S4, Figures S1-S7,
experimental details of DMA pro-drug synthesis, pH-dependent HPLC cleavage
tests, and cell growth inhibition assays. See DOI: 10.1039/x0xx00000x
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Fig. 1 Dox release from DMA prodrug 8 in pH 6.0-7.4 aq. buffer at rt.
DMA 8 improved this ratio to ~ 7:1 (e.g. at 5 h, ~70% of Dox is
released at pH 6 vs. ~10% at pH 7).
Scheme 1 Synthesis of DMA prodrug via DMI precursor.
days. Our initial efforts were focused on using DoxꢀDMAꢀdisulfide
5a/b for further conjugation to the polar, thiolꢀcontaining modulator
glutathione (GSH). However, DMA 5a/b was difficult to handle or
store due to instability. If DMA prodrug is similarly unstable, its
direct practical use is in doubt. To circumvent this difficulty, we
chose to synthesize DoxꢀDMIꢀGSH 7 and then convert it to DMA
prodrug 8 immediately prior to use. Therefore, reaction between Dox
amine and anhydride 4 was allowed to proceed all the way to Doxꢀ
DMIꢀdisulfide 6 with isolated yield of 74%. Disulfide exchange
between 6 and GSH (acid form) went smoothly under acidic
conditions to give DoxꢀDMIꢀGSH 7 in 70% yield. Side products
arising from thiol attacking the maleimide via Michael addition or
ring opening were not observed (Fig. S21 in ESI). On the day DMA
prodrug 8 was used in cleavage tests or cell assays, DMI 7 was
treated with a pH 10 aq. buffer for 1 h at rt to provide the conjugate
base of DoxꢀDMAꢀGSH 8a/b in near quantitative fashion.
To better simulate drug release under in vivo conditions, HPLC
cleavage tests were carried out at 37°C with or without fetal bovine
serum (FBS). As shown in Fig. 2aꢀb (also see Fig. S2 and Table S2
in ESI), in aq. buffer, increasing temperature to 37°C led to faster
Dox release at both pH 6.5 (e.g. ~70% at 3 h) and 7.4 (~20% at 3 h),
yet the pH 6.5 / 7.4 ratio remained ~ 3ꢀ4 fold. When DMA 8 was
exposed to 45% FBS in DMEMꢀF12 cell medium at 37°C, after 6 h,
~60% of Dox was released at pH 6.5 compared to only ~10% at pH
7.4 (Fig. 2cꢀd, also see Fig. S3 and Table S3 in ESI). This 6:1 ratio
largely reflects suppression of Dox release at pH 7.4 (i.e. ~30%
without FBS vs. ~10% with FBS, Fig. 2a vs. 2c) by serumꢀinduced
decomposition of DMA 8 (~25% without FBS vs. ~45% with FBS).
In other words, at pH 7.4, DMA 8 mostly decomposed without
releasing Dox, while at pH 6.5, it mostly cleaved to release Dox.
Thus, the large difference in drug release within the narrow pH range
is accomplished through a combination of ‘switchꢀon’ (i.e. DMA
Subsequently, pH of the prodrug 8 solution was adjusted to a
desired value between 6.0 and 7.4 to evaluate how quickly Dox is
released in response to slight acidity at rt (~25°C). DMA 8
consumption, Dox release, reversion to DMI 7, and background
decomposition were followed over time using HPLC at pH 8 (Fig.
S1 in ESI). At each time point, areas under the peaks were integrated
to give the percentages reported in Table S1 in ESI, which are
plotted in Fig. 1aꢀf. After 24 h at pH 7.4, 19% of prodrug 8 released
Dox while 23% ringꢀclosed to DMI 7 (Fig. 1a). At pH 6.5, drug
release was much faster with 43% of Dox appearing after only 6 h
and 67% after 24 h (Fig. 1d). Overall data of Dox release and DMI 7
formation at different pH values are summarized in Fig. 1gꢀi. Dox
release showed clear pH dependence throughout the pH range
whereas reversion to DMI 7 did not. When drug release is slow at
pH 7.4, ring closure to DMI 7 is competitive, which can serve to
‘switch off’ prodrug over time and reduce background drug release
under physiological pHe. To the best of our knowledge, the largest
pH 6 / 7 cleavage ratio (at 25ºC in aq. buffer) known previously is ~
4:1, achieved by using a spiro diorthoester linker;30 in comparison,
Fig. 2 Dox release from DMA 8 at 37°C with or without serum.
2 | J. Name., 2012, 00, 1-3
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Scheme 2 Potential mechanisms of pH-dependent drug release.
cleavage) and ‘switchꢀoff’ (i.e. DMI formation and other
degradation) mechanisms.
Possible equilibriums that govern pHꢀdependent drug release are
depicted in Scheme 2. DMA acid 8, which is more viable at lower
pH, has at least three modes of reactivity leading to drug release. In
mode A, as proposed by Kirby and Lancaster,1 intramolecular proton
transfer from carboxylic acid to amide followed by ring closure leads
to the tetrahedral intermediate (TI) 10. Breakdown of TI 10 to amine
(Dox) and anhydride 9 completes drug release (this is likely the main
mechanism). The rate of amide hydrolysis is ~ 700ꢀfold faster when
there are two instead of one alkyl substituent on the maleamic acid
double bond,1 as steric congestion of the two alkyl groups force the
reacting carboxyl and amide partners into perfect near attack
conformation.53 Protonation of TI 10 is needed for breakdown,
which may further contribute to the pHꢀdependence of drug release.
Alternatively, ringꢀclosure from DMA conjugate base to 12 may also
lead to Dox release via TI 10 upon protonation. As shown in this
study, considerable amounts of DMI 7 were formed from DMA 8.
Thus, attack on carboxyl by amide N atom must also be possible due
to their close proximity (Scheme 2, Mode C). If so, then similar
attack by amide O atom may also be possible (Scheme 2, Mode B),
which can lead to isomaleimide 13. We are confident that the DMI 7
isolated is not isomaleimide 13 because it is stable under acidic aq.
conditions (pH 2ꢀ3). However, acidꢀcatalyzed hydrolysis of
isomaleimide 13 may also contribute to Dox release via TI 10.
In cancer patients, following intravenous administration of a bolus
dose of Dox (30ꢀ50 mg/m2) over 5ꢀ40 min, the initial plasma
concentration is in the range of 1ꢀ10 µM.54, 55 Dox halfꢀlife in plasma
is only 5ꢀ10 min as the drug is rapidly distributed into tissues.54, 55
Two hours after infusion, the intratumoral concentration of Dox is
mostly in the range of 0.5ꢀ5 µM.54 PK studies suggest that (a) for
Fig. 3 Prodrug 8 can reverse the unfavorable pH-profile of Dox. Cells were
treated with Dox (positive control), Dox-DMA-GSH prodrug 8, Dox-DMI-
GSH 7, or buffer (negative control) under the conditions specified (no
serum except for i), then grown for 2-4 d before counted in viability assays.
Percentages shown are normalized to buffer at pH 7.4. The pH values of
treatment solutions were measured before and after each experiment to
Dox en route to reach its DNA target, the slowest step is transport give the ranges reported. Error bars reflect ± one standard deviation (SD)
(except for (d) they represent 95% confidence level from fitting, see Fig. S7
in ESI). Statistical significances of the difference between the chosen data
sets were evaluated using unpaired (with unequal variance), two-tailed
Student’s t-test (*: P < 0.05; **: P < 0.01; ***: P < 0.001). Exact numerical
across plasmamembrane, which is pHeꢀdependent, (b) peak
intracellular Dox concentration is achieved over a few hours and this
parameter is most critical for antitumor efficacy, and (c) infusion
duration should be 1ꢀ3 h to maximize drug concentration in cancer
cells.49 Our cell experiments were carried out under conditions that
values of the mean, the SD and the P values are reported in ESI Table S4.
reflect these Dox PK parameters. To confirm free drug pHꢀprofile,
Dox was tested in a variety of cancer cells (i.e. ovarian cancer: ESꢀ2,
OVCAR3, SKOV3; breast cancer: MDAꢀMB231; prostate cancer:
PCꢀ3). Indeed Dox was universally more cytotoxic at pHe 7.2ꢀ7.6
than 6.5ꢀ7.0 over a wide range of PKꢀrelevant concentrations (0.1ꢀ4
µM) and time frames (2ꢀ24 h), and often by as much as 2ꢀ3 fold
(Fig. 3, also see ESI Fig. S4ꢀS7).
To test the prodrug, ESꢀ2 cells were treated for 6 h with DMA 8 at
pHe 6.7 or 7.4 (Fig. 3a). Compared to 0.5 µM of free Dox, 1 µM of
prodrug was more potent at pHe 6.7 but ~3ꢀfold less cytotoxic at
pHe 7.4. In terms of pHꢀprofile, the prodrug was ~2ꢀfold more
potent at pHe 6.7 while free drug was the opposite, ~2ꢀfold more
potent at pHe 7.4. This effect of pHꢀprofile reversal diminished
when treatments lasted longer than 6 h (ESI Fig. S6). Compared to
DMA 8, DMI 7 showed much less toxicity (Fig. 3a, also see ESI Fig.
S5) while byproduct
9 was completely not toxic even at
concentration of 200 µM (Fig. S8 in ESI). Next the pHꢀprofile of
prodrug 8 was tested in shorter 2ꢀ3 h experiments, a time frame more
relevant to Dox PK.49 To inhibit the growth of MDAꢀMB231 and
OVCAR3 cancer cells, 3 µM of free Dox was needed; accordingly,
6ꢀ8 µM of prodrug 8 showed 2ꢀ3x higher potency than free drug at
pHe 6.3ꢀ6.4 while being 2ꢀ4x less cytotoxic at pHe 7.5ꢀ7.6 (Fig. 3bꢀ
c). Similar results were also obtained with PCꢀ3 and SKOV3 cells
(ESI Fig. S5). To characterize the pHꢀprofile reversal in a more
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µM. The dose response curves are shown in Fig. S7 in ESI, which
gave prodrug IC50 values of 2.1 ± 0.3 µM at pHe 6.7 and 3.2 ± 0.4
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Financial supports from SUNYꢀBinghamton University and New
York State Department of Health (award 73178 project 1130603 to
M.A. and L.Y.) are gratefully acknowledged. We also thank NSF
MRI grant CHEꢀ0922815 for the 600 MHz NMR, Dr. Juntao Luo
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There are no conflicts to declare.
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DOI: 10.1039/C7CC06843C
TOC entry
Selective drug release at pH 6.7 over pH 7.4 is achieved through a combination of ‘switch-on’
and ‘switch-off’ mechanisms.