Ribonuclease-activated cancer prodrug

Article abstract of DOI:10.1021/ml2002554

Cancer chemotherapeutic agents often have a narrow therapeutic index that challenges the maintenance of a safe and effective dose. Consistent plasma concentrations of a drug can be obtained by using a timed-release prodrug strategy. We reasoned that a ribonucleoside 3′-phosphate could serve as a pro-moiety that also increases the hydrophilicity of a cancer chemotherapeutic agent. Herein, we report an efficient route for the synthesis of the prodrug uridine 3′-(4-hydroxytamoxifen phosphate) (UpHT). UpHT demonstrates timed-released activation kinetics with a half-life of approximately 4 h at the approximate plasma concentration of human pancreatic ribonuclease (RNase 1). MCF-7 breast cancer cells treated with UpHT showed decreased proliferation upon coincubation with RNase 1, consistent with the release of the active drug-4-hydroxytamoxifen. These data demonstrate the utility of a human plasma enzyme as a useful activator of a prodrug.

Full text of DOI:10.1021/ml2002554

Letter
pubs.acs.org/acsmedchemlett
Ribonuclease-Activated Cancer Prodrug
Gregory A. Ellis,†,∥ Nicholas A. McGrath,‡,∥ Michael J. Palte,§ and Ronald T. Raines*,†,‡
‡
§
†Department of Biochemistry, Department of Chemistry, and Medical Scientist Training Program and Molecular & Cellular
Pharmacology Graduate Training Program, University of WisconsinMadison, Madison, Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: Cancer chemotherapeutic agents often have a
narrow therapeutic index that challenges the maintenance of a
safe and effective dose. Consistent plasma concentrations of a
drug can be obtained by using a timed-release prodrug
strategy. We reasoned that a ribonucleoside 3′-phosphate could
serve as a pro-moiety that also increases the hydrophilicity of a
cancer chemotherapeutic agent. Herein, we report an efficient
route for the synthesis of the prodrug uridine 3′-(4-
hydroxytamoxifen phosphate) (UpHT). UpHT demonstrates
timed-released activation kinetics with a half-life of approx-
imately 4 h at the approximate plasma concentration of human pancreatic ribonuclease (RNase 1). MCF-7 breast cancer cells
treated with UpHT showed decreased proliferation upon coincubation with RNase 1, consistent with the release of the active
drug4-hydroxytamoxifen. These data demonstrate the utility of a human plasma enzyme as a useful activator of a prodrug.
KEYWORDS: human pancreatic ribonuclease, 4-hydroxytamoxifen, pharmacokinetics, plasma, tamoxifen, timed-release
any drug candidates have demonstrable therapeutic
potential in vitro but fail in vivo because of poor
Because of the promiscuous activity of ribonucleases, we
reasoned that a chemotherapeutic drug condensed with a
ribonucleoside 3′-phosphate pro-moiety would be released
upon catalysis by RNase 1. We were aware that the use of a
ribonucleoside 3′-phosphate as a pro-moiety would be
facilitated by extant, highly optimized phosphoramidite
chemistry,22,23 making the prodrug readily accessible on a
laboratory or industrial scale. A pendant ribonucleoside 3′-
phosphate could render inactive a small-molecule drug by
hindering the interaction with its target. The hydrophilicity of a
ribonucleoside 3′-phosphate could impart improved pharmaco-
kinetics to hydrophobic drugs.24 Additionally, small molecules
with anionic groups are endowed with reduced rates of
cytosolic uptake and glomerular filtration.25−32
For our proof-of-concept studies, we chose the model parent
drug 4-hydroxytamoxifen (HT). HT is the activated form of
tamoxifen (oxidized by cytochrome P450 enzymes33) and is
significantly more potent than tamoxifen as an antiproliferative
agent against breast cancer cells.34 Tamoxifen acts as an
antiestrogen and is one of the most commonly used hormonal
drugs for the prevention and treatment of breast cancer.35,36
Unfortunately, tamoxifen can have off-target effects and is
linked to an increased risk (2−3%) of endometrial carcinoma
and pulmonary embolism.37 Presumably, these side effects
could be attenuated by delivering tamoxifen at a consistent, low
dose.38−42 Tamoxifen-encapsulated liposomes have been
developed for this purpose,42 but liposomal delivery has, in
general, demonstrated only modest efficacy in the clinic.43
M
pharmacokinetic behavior.1,2 The dosing of chemotherapeutic
agents for cancer, in particular, is made difficult by narrow
therapeutic indices.3,4 Following parenteral administration of a
drug, there is a spike in drug plasma concentration, followed by
a slow decline in concentration as the drug is eliminated or
metabolized, complicating maintenance of the drug at a
beneficial concentration.3,5 Timed-release prodrug technology
provides one potential means to overcome this problem. A pro-
moiety renders the drug inactive until liberation by an enzyme-
catalyzed or nonenzymatic process. Ideally, such timed release
modulates near-toxic peaks or near-ineffective troughs in the
concentration of active drug in plasma.1,3,5−7 Although many
pro-moieties exist,1−7 few provide timed release in plasma.
We sought a pro-moiety that would not only inactivate the
parent drug but also be released during catalysis by an
endogenous plasma enzyme. Fulfilling these criteria is difficult,
as few enzymes have adequate plasma concentrations and many
that do have high specificity for a native substrate. Human
pancreatic ribonuclease (RNase 1;8 EC 3.1.27.5) is an
exception. Contrary to its name, RNase 1 is expressed in
tissues other than pancreas9 and circulates in human plasma at a
concentration of ∼0.4 mg/L.10,11 Moreover, like its renowned
homologue bovine pancreatic ribonuclease (RNase A12,13),
RNase 1 catalyzes the cleavage of RNA by a transphosphor-
ylation reaction14−16 and has little specificity for its leaving
group.17−21 This promiscuity is the basis for the tumor-targeted
activation of a phenolic nitrogen mustard from a ribonucleoside
3′-phosphate prodrug using an antibody−RNase 1 variant in an
antibody-directed enzyme prodrug therapy (ADEPT) strat-
egy.21
Received: October 25, 2011
Accepted: February 28, 2012
Published: February 28, 2012
© 2012 American Chemical Society
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Letter
Hence, we elected to attach HT to uridine 3′-phosphate and
analyze the activation of this model prodrug by RNase 1
(Figure 1).
Scheme 1. Synthesis of UpHT
Figure 1. Scheme showing the cleavage of prodrug UpHT by RNase 1
to yield uridine 2′,3′-cyclic phosphate (U>p) and HT.
Uridine 3′-(4-hydroxytamoxifen phosphate) (UpHT) was
synthesized in five steps from commercially available HT
(≥70% Z isomer, which is the more active form44,45) and
uridine phosphoramidite (Scheme 1). Briefly, HT was coupled
to uridine phosphoramidite by using N-methylbenzimidazolium
triflate as a catalyst.46 The coupled product was oxidized with
iodine and deprotected stepwise. The final product was purified
by reverse-phase HPLC on C18 resin to provide UpHT in an
overall yield of 58%.
We expected the uridine 3′-phosphate moiety of UpHT to
endow the prodrug with greater hydrophilicity than the parent
drug, which could improve pharmacokinetic behavior. To
investigate this issue, we calculated the partition (log P) and
distribution (log D) coefficients of UpHT and HT.47 The
calculated log P and log D values of UpHT were indeed
significantly lower than those of the parent drug HT (Table 1),
indicative of increased hydrophilicity.
To be the basis for an effective timed-release prodrug
strategy, the pro-moiety needs to be released by the activating
enzyme over time. Hence, we assessed the RNase 1-catalyzed
rate of HT-release from UpHT. To do so, RNase 1 (final
concentration: ∼0.15 μg/mL) was added to 0.10 M 2-(N-
morpholino)ethanesulfonic acid (MES)−NaOH buffer, pH 6.0,
containing NaCl (0.10 M) and UpHT (0.090 mM).48−50 The
reaction mixture was incubated at 37 °C, and aliquots were
withdrawn at known times and assayed for HT by HPLC.
Under these conditions, which are typical for assays of
ribonucleolytic activity,12,48 HT was released with a half-life
of ∼4 h (Figure 2). Importantly, UpHT was stable in the
absence of RNase 1; after 11 h at 37 °C, <6% of UpHT had
degraded to HT.
Table 1. Calculated Partition and Distribution Coefficients
of HT and UpHT47
coefficient
HT
UpHT
log P (nonionized)
log P (ionized)
6.05
2.55
4.66
5.69
3.88
−2.00
0.12
log D (pH = 7.4)
a
log D (pH = pI )
−1.79
a
HT, pI = 9.01; UpHT, pI = 5.00.
validate that UpHT is inherently unstable at pH 7.4 (as
opposed to the medium containing contaminating ribonu-
cleases), the stability of UpHT was assessed in ribonuclease-
free 0.10 M sodium phosphate buffer, pH 7.4, containing NaCl
(0.10 M). Again, the half-life was ∼9 h. The instability of
UpHT at pH 7.4 is consistent with HT being a good leaving
group, as its hydroxyl group has pKa ∼9.3.47 By comparison, the
P−O5′ bond in RNA has a half-life of 4 years.52
To demonstrate the efficacy of UpHT in cellulo, we
monitored its effect on the proliferation of MCF-7 breast
cancer cells, which are known to be vulnerable to HT.34 UpHT
was made more antiproliferative by the presence of added
To assess the unmasking of UpHT under more physiological
conditions, HT release from UpHT was monitored in cell
culture medium (Figure 3A). In medium without added
ribonucleases, HT was released with a half-life of ∼9 h. To
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ACS Medicinal Chemistry Letters
Letter
a self-immolative linker to an amino group.4 RNase 1 is known
to cleave RNA faster after pyrimidine than purine nucleo-
bases.53 Hence, cytidine- and uridine-masked drugs are likely to
be activated more rapidly than adenosine- and guanosine-
masked drugs. In addition, synergistic drugs could be
conjugated to different ribonucleoside 3′-phosphates to achieve
simultaneous release of drugs at desired concentrations. These
same effects could be used to optimize simultaneous plasma
concentrations of chemoprotective drugs and chemotherapeu-
tic drugs. The pharmacokinetics of the drug could be tuned
further by modification of the ribose 5′-hydroxyl group. For
instance, this hydroxyl group could be PEGylated to enhance
serum half-life, extended with additional nucleoside 3′-
phosphates to increase hydrophilicity, or alkylated with the
intent of increasing hydrophobicity.54,55
Figure 2. Progress curve for the release of HT from UpHT (0.090
mM) by RNase 1 (∼0.15 μg/mL) in 0.10 M MES−NaOH buffer, pH
6.0, containing NaCl (0.10 M) at 37 °C. Inset: t < 1 h.
Finally, we note that nucleoside 3′-phosphate pro-moieties
could impart selective activation of chemotherapeutic agents
near tumor sites. Although RNase 1 was employed herein due
to its abundance in plasma,8,9,56,57 RNase 1 homologues might
also activate prodrugs like UpHT in situ.8 One such homologue
is eosinophil-derived neurotoxin (RNase 2), which is carried
and released by eosinophils.8 These cells are known to
accumulate and degranulate at tumor sites.58−60 We anticipate
that, akin to prodrug monotherapy (PMT) in which prodrugs
are activated by endogenous enzymes found in abundance near
tumors,61 a prodrug strategy reliant on RNase 2 could be used
to generate active drugs at adventitious sites. Studies to probe
the versatility of the RNase 1/ribonucleoside 3′-phosphate
prodrug system are underway in our laboratory.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data and experimental protocols. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 608-262-8588. E-mail: rtraines@wisc.edu.
Author Contributions
Figure 3. Stability of UpHT and effect of UpHT on the proliferation
of MCF-7 cells. All data points are the means ( SEs) of separate
experiments carried out in triplicate. (A) Progress curves for the
release of HT from UpHT (40 μM) at 37 °C in ribonuclease-free 0.10
∥These authors contributed equally to this work.
Funding
This work was supported by Grant R01 CA073808 (NIH).
N.A.M. was supported by the postdoctoral fellowship F32
GM096712 (NIH). M.J.P. was supported by Molecular and
Cellular Pharmacology Training Grant T32 GM008688 (NIH)
and predoctoral fellowship 09PRE2260125 (American Heart
Association). This study made use of the National Magnetic
Resonance Facility at Madison, which is supported by NIH
Grants P41RR02301 (BRTP/NCRR) and P41GM66326
(NIGMS). Additional equipment was purchased with funds
from the University of Wisconsin, the NIH (RR02781,
RR08438), the NSF (DMB-8415048, OIA-9977486, BIR-
9214394), and the U.S. Department of Agriculture.
△
M sodium phosphate buffer, pH 7.4, containing NaCl (0.10 M) (
;
t1/2 = 9.4 h) and serum-free51 medium in the absence ( ; t1/2 = 9.0 h)
○
●
and presence ( ; t1/2 = 4.4 h) of RNase 1 (0.4 μg/mL). (B)
Proliferation of MCF-7 cells in serum-free51 medium, monitored by
the incorporation of [methyl-3H]thymidine into cellular DNA. UpHT
○
●
□
in the absence ( ; IC50 = 16.7 0.8 μM) and presence ( ; IC50 = 5.2
0.2 μM) of RNase 1 (6.2 μg/mL). HT in the absence ( ; IC50 = 2.7
0.1 μM) and presence ( ; IC50 = 2.7 0.4 μM) of RNase 1.
■
RNase 1 (Figure 3B), indicating that UpHT is a ribonuclease-
activatable prodrug. Thus, we have demonstrated proof-of-
concept for a prodrug strategy that employs a human plasma
enzyme to release a cancer chemotherapeutic agent in a timed-
release manner (Figure 1).
In addition to the attributes evident in UpHT, the RNase 1/
ribonucleoside 3′-phosphate prodrug system has versatile
modularity. For example, the leaving group need not be an
aryloxy group. Pancreatic-type ribonucleases catalyze the
cleavage of P−O bonds to alkoxy groups, which could include
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge M. L. Hornung for early contributions to this
work.
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ABBREVIATIONS
■
HT, hydroxytamoxifen; MES, 2-(N-morpholino)ethanesulfonic
acid; RNase 1, human pancreatic ribonuclease; U>p, uridine
2′,3′-cyclic phosphate; UpHT, uridine 3′-(4-hydroxytamoxifen
phosphate)
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