Optimization of an albumin-binding prodrug of doxorubicin that is cleaved by prostate-specific antigen

Article abstract of DOI:10.1021/ml100060m

We have developed a novel albumin-binding prodrug of doxorubicin that incorporates p-aminobenzyloxycarbonyl (PABC) as a 1,6 self-immolative spacer in addition to the heptapeptide, Arg-Ser-Ser-Tyr-Tyr-Ser-Leu, as a substrate for the prostate-specific antigen (PSA) that is overexpressed in prostate carcinoma and represents a molecular target for selectively releasing an anticancer agent from a prodrug formulation. The prodrug exhibited good water solubility and was bound rapidly to the cysteine-34 position of human serum albumin. Incubation studies with PSA demonstrated that the albumin-bound form of the prodrug was cleaved rapidly at the P₁?P₁? scissile bond, releasing H-Ser-Leu-PABC-DOXO, which was further degraded to release doxorubicin as a final cleavage product within a few hours in prostate tumor tissue homogenates as well as in PSA-positive LNCaP LN cell lysates. Moreover, our prodrug exhibited antiproliferative activity in a low micromolar range against a PSA-expressing prostate cancer cell line (LNCaP).

Full text of DOI:10.1021/ml100060m

pubs.acs.org/acsmedchemlett
Optimization of an Albumin-Binding Prodrug of
Doxorubicin That Is Cleaved by Prostate-Specific Antigen
‡
†
†
§
Bakheet Elsadek,†, Ralph Graeser, Andre Warnecke, Clemens Unger, Tahia Saleem,
ꢀ
Nagla El-Melegy,§ Hafez Madkor, and Felix Kratz*,†
†Tumor Biology Center, Division of Macromolecular Prodrugs, Breisacher Strasse 117, 79106 Freiburg, Germany,
‡ProQinase GmbH, Breisacher Strasse 117, 79106 Freiburg, Germany, §Department of Biochemistry, Faculty of Medicine,
Assiut University, P.O. Box No. 71526, Assiut, Egypt, and Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University,
P.O. Box No. 71524, Assiut, Egypt
ABSTRACT We have developed a novel albumin-binding prodrug of doxorubicin
that incorporates p-aminobenzyloxycarbonyl (PABC) as a 1,6 self-immolative
spacer in addition to the heptapeptide, Arg-Ser-Ser-Tyr-Tyr-Ser-Leu, as a substrate
for the prostate-specific antigen (PSA) that is overexpressed in prostate carcinoma
and represents a molecular target for selectively releasing an anticancer agent from
a prodrug formulation. The prodrug exhibited good water solubility and was bound
rapidly to the cysteine-34 position of human serum albumin. Incubation studies
with PSA demonstrated that the albumin-bound form of the prodrug was cleaved
0
rapidly at the P1-P1 scissile bond, releasing H-Ser-Leu-PABC-DOXO, which was
further degraded to release doxorubicin as a final cleavage product within a few
hours in prostate tumor tissue homogenates as well as in PSA-positive LNCaP LN
cell lysates. Moreover, our prodrug exhibited antiproliferative activity in a low
micromolar range against a PSA-expressing prostate cancer cell line (LNCaP).
KEYWORDS Doxorubicin, albumin, prodrug, drug delivery, prostate cancer, pros-
tate-specific antigen
rostate cancer is the most common nondermatologi-
thiol-binding prodrug to the cysteine-34 position of circulat-
ing albumin after intravenous administration with subse-
quent accumulation of the drug-albumin conjugate in the
tumor due to passive targeting and (2) release of the
albumin-bound drug at the tumor site due to the incorpora-
tion of a cleavable bond between the drug and the carrier.
The first and most advanced prototype of these types of
prodrugs is the (6-maleimidocaproyl)hydrazone derivative
of doxorubicin (DOXO-EMCH, now INNO-206), an acid-
sensitive prodrug of doxorubicin that is under phase II
clinical development9 (see http://www.cytrx.com). Inspired
by the translational research with DOXO-EMCH, a broad
spectrum of albumin-binding prodrugs has been developed
by Kratz and co-workers that incorporates an enzymatically
cleavable peptide linker. Examples include doxorubicin pro-
drugs that are cleaved by matrix metalloproteases 2 and 9,8
cathepsin B,10 urokinase,11 or prostate-specific antigen
(PSA),12,13 methotrexate prodrugs that are cleaved by cathe-
psin B and plasmin,14 and camptothecin prodrugs that are
cleaved by cathepsin B.10
cal male malignancy and is the second most common
1
Pcause of male cancer-related death. Prostate cancer
progression is characterized by metastases primarily in the
bone and lymph nodes, and to date, there is no effective
therapy for treating patients with metastatic disease if they
no longer respond to hormone therapy.2,3 The benefit of
therapy with conventional chemotherapy is limited due to
the systemic toxicity and lack of tumor selectivity of anti-
neoplastic agents. Thus, there is an urgent medical need to
design drug delivery systems that transport the anticancer
agent to the primary tumor and metastases.
Besides the development of suitable galenic formulations
such as liposomes or micelles, several promising carrier-
linked prodrug approaches have been developed over the
last decades that rely on attaching chemical groups or
carriers to the drug through a predetermined cleavage point
that is specifically cleaved to release the drug in the tumor
cell or tumor tissue, either extra- or intracellularly.4 A drug
carrier that is playing an increasing role in the clinical setting
is human serum albumin (HSA) (66.5 kDa).5 Uptake of
albumin in tumors is mediated by the enhanced permeabil-
ity and retention (EPR) effect, that is, EPR of macromole-
cules in relation to passive tumor targeting (reviewed in ref 6).
Over the past years, we have investigated a targeting strat-
egy that is based on two features:5,7,8 (1) in situ binding of a
Among these proteases, the PSA is especially attractive as
a target protease because it is almost exclusively expressed
Received Date: March 25, 2010
Accepted Date: May 3, 2010
Published on Web Date: May 26, 2010
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dipeptide by incorporating a self-eliminating linker between
Arg and doxorubicin. Arg was initially chosen in this position
because our previous studies with doxorubicin amino acid
derivatives had shown that Arg-DOXO was cleaved more
efficiently in tumor homogentates than other doxorubicin
amino acid derivatives.11 As a self-eliminating linker, we
selected p-aminobenzyloxycarbonyl (PABC), which was in-
itially introduced by Carl et al.22 and proved to reduce a
possible steric hindrance of bulky drugs during the enzy-
matic cleavage process.23 Upon deacylation, the PABC
spacer decomposes in a 1,6-benzyl elimination and sponta-
neously releases the free drug.22
Unfortunately, our synthetic efforts to obtain H-Ser-Arg-
PABC-DOXO failed as a result of the chemical incompatibility
of the Arg moiety in this position. Alternatively, we synthe-
sized the doxorubicin dipeptide H-Ser-Leu-PABC-DOXO (6).
Leucine is known to occupy this position in some natural PSA
peptide substrates24 and has also been used by the Merck
group in the development of their low molecular weight
doxorubicin prodrug L-377,202 Mu-His-Ser-Ser-Lys-Leu-Gln-
Leu-DOXO (Mu = morpholinocarbonyl).20
Compound 6 could be synthesized in good yields, and as a
comparison, we also synthesized the doxorubicin-dipeptide
H-Ser-Leu-DOXO (8) (see the Supporting Information) and
subsequently compared theircleavage profiles. Compound 6
showed a clearly improved cleavage profile over 8 after
incubation with LNCaP tumor homogenates (Figure 2a) as
well as in PSA-positive LNCaP cell lysates (Figure 2b), releas-
ing doxorubicin as the final cleavage product within a few
hours. In contrast, 8 was degraded slowly in the LNCaP
tumor homogenates (Figure 3a) as well as in the PSA-positive
LNCaP cell lysates (Figure 3b), being cleaved to the doxo-
rubicin monopeptide H-Leu-DOXO (7) that slowly liberated a
minimal amount of doxorubicin over 20 h.
In a similar way, H-Ser-Arg-DOXO was reported to release
the doxorubicin monopeptide H-Arg-DOXO as a final clea-
vage product, which liberated a small percent of doxorubicin
over 24 h in the LNCaP tumor homogenates.13 These results
underline the pivotal role of the PABC spacer for ensuring
effective degradation of suitable doxorubicin dipeptide deri-
vatives to doxorubicin, which is apparently catalyzed by
unspecified proteases in tumor homogenates. In subsequent
cell culture experiments using the PSA-expressing prostate
cancer cell line LNCaP [transduced with a luciferase-neomy-
cin (LN) resistance fusion gene cassette, which allows for
sensitive quantification of viable cells with a wide dynamic
range], the cytotoxicity of the different doxorubicin dipep-
tides, 6, 8, and H-Ser-Arg-DOXO correlated well with their
cleavage rates (see Table 1).
Figure 1. Structure of the PSA-cleavable prodrug PSA5.13
in prostate tissue and prostate carcinoma. PSA is a serine
protease that belongs to the kallikrein gene family with
chymotrypsin-like activity that is involved in the hydrolytic
processing of semenogelins (cleavage of the seminal fluid
proteins semenogelin I and II), which is required for lique-
faction of seminal fluids.15 Overexpression of PSA has
primarily been demonstrated in prostate carcinoma and at
low levels in breast cancer.16-18 PSA levels in human pri-
mary prostate tumors can be very high depending on the
stage of the disease (up to 1600-2100 nM), and 80-90% of
the secreted PSA is active in the tumor environment.17
PSA is secreted in an active form in prostate cancer but
forms two stable complexes with R1-antichymotrypsin and
R2-macroglobulin in the blood. PSA complexed to R1-antic-
hymotrypsin is the predominant fraction of PSA. Only a
minor fraction of serum PSA is not associated with protei-
nase inhibitors, and it is unknown whether this free fraction
still has enzymatic activity.18 In addition, it has recently
been shown that PSA in mice bearing LNCaP tumors forms
complexes similar to those in man, but the major immu-
noreactive complex contains R1-antitrypsin rather than
R1-antichymotrypsin.19
Consequently, we and others have investigated prodrug
strategies that exploit PSA as a molecular target for releasing
an anticancer drug in prostate tumors.12,13,19-21 Recently, a
PSA-cleavable albumin-binding prodrug of doxorubicin
EMC-Arg-Ser-Ser-Tyr-Tyr-Ser-Arg-DOXO (abbreviated PSA5;
EMC = ε-maleimidocaproic acid) was developed in our
group and is depicted in Figure 1.13
PSA5 was rapidly bound to circulating albumin and was
superior over doxorubicin in an orthotopic PSA-positive
model (LNCaP) with respect to antitumor efficacy and toler-
ability but did not induce tumor remissions.13 Cleavage
studies of the albumin-bound form of PSA5 (HSA-PSA5) with
PSA demonstrated an efficient cleavage between Tyr and
Ser, releasing the doxorubicin dipeptide H-Ser-Arg-DOXO as
a final cleavage product within 24 h. This dipeptide, however,
was cleaved slowly to H-Arg-DOXO in LNCaP tumor homo-
genates, and only minute amounts of doxorubicin were
released. These results revealed that the full potential of
the prodrug had not been exploited considering that only
small amounts of the active agent doxorubicin were liber-
ated in PSA-positive prostate carcinoma tissue.13
Compound 6, which rapidlyproduced doxorubicin in LNCaP
cell lysates, exhibited an IC50 value in the low micromolar
range of 0.15 μM that was quite comparable to the cytotoxi-
city of doxorubicin, which had an IC50 value of 0.11 μM
(Table 1). On the other hand, both of the doxorubicin
dipeptides lacking the PABC self-eliminating linker were
either inactive against the PSA-positive LNCaP LN cells as
in the case of H-Ser-Arg-DOXO with an IC50 value of >50 μM
or proved to be approximately 20-fold less active (IC50 value
of 3.09 μM for 8).
As a consequence and as a main goal of the present work,
we set out to optimize the release characteristics of PSA-
cleavable prodrugs with albumin-binding properties that
would rapidly liberate doxorubicin as the final cleavage
product. Initially, we aimed to synthesize and subsequently
investigate the in vitro cytotoxicity of a new doxorubicin
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Figure 2. Chromatograms of incubation studies of 6 (50 μM) in the presence of (a) LNCaP tumor tissue homogenates and (b) LNCaP cell
lysates at 37 °C.
Figure 3. Chromatograms of incubation studies of 8 (50 μM) in the presence of (a) LNCaP tumor tissue homogenates and (b) LNCaP cell
lysates at 37 °C.
These encouraging results prompted us to synthesize a
novel albumin-binding prodrug of doxorubicin that incorpo-
rates the PABC self-immolative spacer between the doxor-
ubicin and the peptide substrate. The new prodrug EMC-
Arg-Ser-Ser-Tyr-Tyr-Ser-Leu-PABC-DOXO (9) was obtained
through coupling of 6 with EMC-Arg-Ser-Ser-Tyr-Tyr-OH in
anhydrous dimethyl formamide (DMF) in the presence of 1-
hydroxybenzotriazole hydrate (HOBt), 4-methylmorpholine,
and N,N0-diisopropylcarbodiimide (DIPC) (see Supporting
Information - Scheme 1S).
The prodrug 9 was purified on a C-18 reverse-phase
column. The lyophilized red product was characterized by
mass spectrometry, and its purity was determined by HPLC
(see Supporting Information). Compound 9 exhibited good
water solubility (∼4 mg/mL in isotonic 5% glucose solution)
and was bound rapidly within a few minutes to the cysteine-
34 position of endogenous albumin in a Michael addition in
accordance to our previous work on the albumin binding
properties of maleimide-bearing prodrugs (see the Support-
ing Information).
Table 1. IC50 Values of Doxorubicin, 6, 8, HSA-9, and HSA-PSA-5 in
the LNCaP LN Cell Line and the p Values of These IC50 Values in
Correlation to That of Doxorubicin
compounds
IC50 value (μM)
p values
doxorubicin
0.11 ( 0.008
33.78 ( 12
1.30 ( 0.15
0.15 ( 0.03
3.09 ( 0.86
>50.0
HSA-PSA-5
0.0073
0.0002
0.7600
0.0035
NDa
HSA-9
6
8
H-Ser-Arg-DOXO
a ND, not determined.
0
(HSA-9) was rapidly cleaved at the P1-P1 scissile bond,
releasing 6 as the cleavage product within 3 h (Figure 4a).
Compound 6 was also observed as an intermediate clea-
vage product in an incubation study of HSA-9 over 6 h in the
LNCaP tumor homogenate as detected by HPLC, but free
doxorubicin was the predominant final cleavage product
(Figure 4b). The overall reactions that are involved in the
cleavage process of HSA-9 are depicted in Scheme 1. In-
cubation studies of HSA-9 with PSA showed complete clea-
vage to 6 after 3 h under the experimental conditions chosen
(20 μg/mL PSA; Figure 4a), and complete cleavage to
In addition, the prodrug showed a good stability in human
plasma as well as in the PSA cleavage buffer (Tris buffer, pH
7.8) over 24 h (Figure 4a). Incubation studies with PSA
demonstrated that the albumin-bound form of the prodrug
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Figure 4. Chromatograms of incubation studies of HSA-9 in the presence of (a) enzymatically active human PSA (20 μg/mL) and (b) LNCaP
tumor tissue homogenates at 37 °C.
Scheme 1. Schematic Illustration of the Binding of Prodrug 9 to the Cysteine-34 Position of Albumin and Subsequent Enzymatic Cleavage
by PSA of the Formed Albumin Conjugate and Degradation of 6 to Doxorubicin by Further Proteases
doxorubicin was observed after incubation of HSA-9 with a
homogenate from a LNCaP tumor after 6 h (see Figure 4b).
This cleavage pattern can be judged as a significant improve-
ment over the former HSA-PSA5, which only yielded small
amounts of Arg-DOXO and doxorubicin when incubated
with LNCaP tumor homogenates.13
value of 1.3 μM against LNCaP cells (see Table 1). Consistent
with our earlier results with PSA-cleavable doxorubicin
prodrugs,12,13 HSA-9 possesses a significantly higher IC50
value than doxorubicin, presumably due to the low PSA
levels secreted in the cell culture medium during 96 h
(26.9-312 ng/mL PSA; see the Supporting Information).
Of note is that HSA-9 was approximately 26-fold more active
than HSA-PSA5 against the PSA-positive LNCaP LN cells,
which can be explained by a more efficient cleavage of
HSA-9 by PSA with concomitant liberation of doxorubicin,
which is far more pronounced than for HSA-PSA5.
In conclusion, by introducing the self-eliminating spacer
PABC, we have optimized the cleavage profile as well as the
release characteristics of a PSA-cleavable prodrug with albu-
min-binding properties that rapidly liberate doxorubicin as
the final cleavage product. In addition, in stability studies of
HSA-9 with murine serum from the experiment in the LNCAP
model that contains a high PSA level from a control tumor-
bearing animal (PSA ∼ 150 ng/mL) as well as with human
serum from a patient with prostate cancer that showed a
very high level of circulating PSA in his serum (PSA ∼718 ng/mL),
only marginal cleavage of doxorubicin or other byproduct
was observed over 24 h at 37 °C (data not shown), making it
a worthy candidate for further preclinical studies.
To clarify the role of PSA is the cleavage of HSA-9, the
chromatogram in Figure 4b in the background shows a cleavage
study of HSA-9 with a homogenate of a healthy murine prostate
over 24 h. There is no indication of the appearance of 6, andonly
small amounts of doxorubicin are liberated over this long time
period. Furthermore, we have performed cleavage studies
of HSA-9 with a PSA-negative prostate tumor homogenate
(xenograft DU145). The homogenate was prepared identically
to the LNCaP tumor (200 mg of tumor in 800 μL of Tris buffer,
pH 7.8). The chromatograms over 24 h are shown in the
Supporting Information (Figure 3S). Only marginal cleavage
to 6 and doxorubicin was observed after 16-24 h. These
comparative results clearly indicate that PSA is involved in the
cleavage of HSA-9because incubation studies with homogenates
from LNCaP tumor homogenates showed 6 as an intermediate
and complete cleavage to doxorubicin already after 6 h.
In the cell culture experiments, HSA-9 exhibited antipro-
liferative activity in the low micromolar range with an IC50
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We have recently completed an in vivo study with the new
PSA-cleavable prodrug 9 in the orthotopic LNCaP model, and
9 demonstrated a distinct superiority over doxorubicin and
the former doxorubicin prodrug lacking the PABC spacer
(PSA5), especially regarding the antitumor effects on lung
and bone metastases. We will report on these results in a
separate communication.25
(11) Chung, D. E.; Kratz, F. Development of a novel albumin-
binding prodrug that is cleaved by urokinase-type-plasmino-
gen activator (uPA). Bioorg. Med. Chem. Lett. 2006, 16, 5157–
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(12) Kratz, F.; Mansour, A.; Soltau, J.; Warnecke, A.; Fichtner, I.;
Unger, C.; Drevs, J. Development of albumin-binding doxor-
ubicin prodrugs that are cleaved by prostate-specific antigen
(PSA). Arch. Pharm. 2005, 338, 462–472.
(13) Graeser, R.; Chung, D. E.; Esser, N.; Moor, S.; Schachtele, C.;
Unger, C.; Kratz, F. Synthesis and biological evaluation of an
albumin-binding prodrug of doxorubicin that is cleaved by
prostate-specific antigen (PSA) in a PSA-positive orthotopic
prostate carcinoma model (LNCaP). Int. J. Cancer 2008, 122,
1145–1154.
SUPPORTING INFORMATION AVAILABLE Procedures for
the preparation of the key compounds 6, 8, and 9 and their mass
spectra as well as the methods and information on binding,
cleavage, and cytotoxicity studies. This material is available free
of charge via the Internet at http://pubs.acs.org.
(14) Warnecke, A.; Fichtner, I.; Sass, G.; Kratz, F. Synthesis,
cleavage profile, and antitumor efficacy of an albumin-bind-
ing prodrug of methotrexate that is cleaved by plasmin and
cathepsin B. Arch. Pharm. (Weinheim) 2007, 340, 389–395.
(15) Akiyama, K.; Nakamura, T.; Iwanaga, S.; Hara, M. The
chymotrypsin-like activity of human prostate-specific anti-
gen, gamma-seminoprotein. FEBS Lett. 1987, 225, 168–172.
(16) Diamandis, E. P. New diagnostic applications and physiolo-
gical functions of prostate specific antigen. Scand. J. Clin. Lab.
Invest. Suppl. 1995, 221, 105–112.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: þ49-761-2062930. Fax: þ49-761-2062905. E-mail:
kratz@tumorbio.uni-freiburg.de.
Funding Sources: We gratefully thank the Dietmar Hopp Foun-
dation and the Egyptian Ministry of Higher Education for financial
support.
(17) Denmeade, S. R.; Sokoll, L. J.; Chan, D. W.; Khan, S. R.; Isaacs,
J. T. Concentration of enzymatically active prostate-specific
antigen (PSA) in the extracellular fluid of primary human
prostate cancers and human prostate cancer xenograft
models. Prostate 2001, 48, 1–6.
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gen ratio;Hope and controversies. Eur. Urol. 1997, 31, 385–
388.
(19) Denmeade, S. R.; Isaacs, J. T. Enzymatic activation of pro-
drugs by prostate-specific antigen: Targeted therapy for meta-
static prostate cancer. Cancer J. Sci. Am. 1998, 4 (Suppl. 1),
S15–S21.
(20) DeFeo-Jones, D.; Garsky, V. M.; Wong, B. K.; Feng, D. M.;
Bolyar, T.; Haskell, K.; Kiefer, D. M.; Leander, K.; McAvoy, E.;
Lumma, P.; Wai, J.; Senderak, E. T.; Motzel, S. L.; Keenan, K.;
Van Zwieten, M.; Lin, J. H.; Freidinger, R.; Huff, J.; Oliff, A.;
Jones, R. E. A peptide-doxorubicin 'prodrug' activated by
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