Synthesis, cleavage profile, and antitumor efficacy of an albumin-binding prodrug of methotrexate that is cleaved by plasmin and cathepsin B

Article abstract of DOI:10.1002/ardp.200700025

Cathepsin B and plasmin are intra- or extracellular proteases that are overexpressed by several solid tumors. In order to exploit both proteases as molecular targets for tumor-specific cleavage of prodrugs, an albumin-binding formulation of methotrexate w

Full text of DOI:10.1002/ardp.200700025

Arch. Pharm. Chem. Life Sci. 2007, 340, 389–395
A. Warnecke et al.
389
Full Paper
Synthesis, Cleavage Profile, and Antitumor Efficacy of an
Albumin-Binding Prodrug of Methotrexate that is Cleaved by
Plasmin and Cathepsin B
Andrꢀ Warnecke1, Iduna Fichtner2, Gretel Saß3, and Felix Kratz1
1 Tumor Biology Center, Freiburg, Germany
2 Max-Delbrꢀck Center, Berlin, Germany
3 medac GmbH, Wedel, Germany
Cathepsin B and plasmin are intra- or extracellular proteases that are overexpressed by several
solid tumors. In order to exploit both proteases as molecular targets for tumor-specific cleavage
of prodrugs, an albumin-binding formulation of methotrexate was developed that incorporated
the peptide sequence D-Ala-Phe-Lys as the protease substrate. Albumin is a suitable carrier for
cytostatic agents due to passive accumulation in solid tumors. Synthesis was performed by cou-
pling the peptide linker EMC-D-Ala-Phe-Lys(Boc)-Lys-OH (EMC = e-maleimidocaproic acid) to the c-
COOH group of a-tert-butyl protected methotrexate. After cleavage of the protective groups and
purification on reverse phase HPLC, a highly water-soluble methotrexate-peptide derivative was
obtained that binds rapidly and selectively to human serum albumin. The albumin-bound form
of the prodrug was shown to be efficiently cleaved by cathepsin B and plasmin as well as in an
ovarian carcinoma homogenate (OVCAR-3) liberating a methotrexate-lysine derivative. In an
OVCAR-3 xenograft model, the prodrug at a dose of 4615 mg/kg methotrexate equivalents dem-
onstrated distinctly superior antitumor efficacy compared to free methotrexate at a dose of
46100 mg/kg [T/C(%) for MTX = 69; T/C(%) for MTX prodrug = 29]. The data provide a further
proof of concept for the development of albumin-binding, enzymatically cleavable prodrugs of
anticancer drugs.
Keywords: Cathepsin B / Human serum albumin / Macromolecular prodrug / Methotrexate / Plasmin /
Received: February 12, 2007; accepted: May 4, 2007
DOI 10.1002/ardp.200700025
iting disorders. When administered, most low-molecular
weight anticancer agents are evenly distributed over the
organism thus damaging all proliferating cells including
healthy ones. Therefore, therapeutic strategies to over-
come these drawbacks and to make anticancer drugs act
more tumor-specifically should concentrate on inherent
differences between normal and malignant tissue. A
promising approach is to employ macromolecules as
vehicles for anticancer drugs [1]. One to several low-
molecular weight drugs are covalently linked to a macro-
molecular carrier, e.g. synthetic polymers or serum pro-
teins. This macromolecular prodrug concept exploits the
differences between tumor tissue and healthy tissue on
two levels: First, a passive accumulation of macromole-
cules in tumor tissue is achieved due to vascular anoma-
Introduction
Therapy of cancer diseases with cytostatic drugs is
mainly restricted by adverse effects that range from cos-
metic problems to severe and – consequently – dose-lim-
Correspondence: Dr. Felix Kratz, Tumor Biology Center, Department of
Medical Oncology, Clinical Research, Breisacher Straße 117, D-79106
Freiburg, Germany.
E-mail: felix@tumorbio.uni-freiburg.de
Fax: +49 761 206-2905
Abbreviations: Boc: tert-butoxycarbonyl; BOP: (benzotriazol-1-yloxy)-
tris-(dimethylamino)phosphonium hexafluorophosphate; DIEA: diisopro-
pylethylamine; DMF: N,N-dimethylformamide; HATU: O-(7-azabenzotria-
zol-1-yl)-N,N,N9,N9-tetramethyluronium hexafluorophosphate; HSA: hu-
man serum albumin; MTD: maximum tolerated dose; MTX: methotrexate
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Arch. Pharm. Chem. Life Sci. 2007, 340, 389–395
lies and an impaired lymphatic drainage (EPR, enhanced
permeability and retention) [2]. Second, enzymes that are
overexpressed in malignant tissue can be exploited for
tumor-specific release of the carrier-bound drugs.
The overexpression of a variety of proteases is well
documented for a number of different tumors, and these
enzymes are known to play a decisive role in metastasis
and tumor progression [3–5]. Elevated levels were
reported for cathepsins [6], matrix metalloproteases [7],
urokinase-type plasminogen activator (uPA) [8], prostate-
specific antigen (PSA) [9], and plasmin [8]. Short peptide
sequences that are cleaved by these proteases can be
employed as linkers acting as a molecular bridge
between the drug and the carrier. Ideally, such linkers
are non-toxic and non-immunogenic, plasma stable, and
rapidly and selectively cleaved in malignant tissue to
release the drug in its active form.
Figure 1. Chromatogram of human plasma, 3, and plasma incu-
bated with 3.
Previously, we developed a number of albumin-bind-
ing prodrugs of doxorubicin incorporating peptide
sequences that were cleaved by uPA [10], PSA [11], cathe-
psin B [12], and the matrix metalloproteases 2 and 9
[13, 14]. These prodrugs were shown to bind rapidly and
selectively to circulating serum albumin after intrave-
nous administration, and the respective drug-albumin
conjugates were selectively cleaved by the target
enzymes or after incubation with native tumor material
(tissue homogenate). In this work, we report on the first
albumin-binding formulation of the anticancer drug
methotrexate (MTX). The prodrug incorporates the pep-
tide sequence D-Ala-Phe-Lys which is known to be cleaved
by either the extracellular enzyme plasmin [15] or the
lysosomal protease cathepsin B [16]. For both proteases,
an overexpression in various solid tumors is documented
[6, 8]. Synthesis of the prodrug, HPLC studies on stability
in plasma and its cleavage properties in the presence of
the target enzymes as well as an in vivo experiment in
xenografted mice are described.
with the preparation of a-protected MTX (2). Compound 2
was obtained by condensing the MTX precursor 4-amino-
4-deoxy-N10-methylpteroic acid (APA) with H-Glu(OMe)-Ot-
Bu using BOP as the coupling agent (see Scheme 1). Subse-
quent hydrolysis of the methyl ester 1 afforded the MTX
a-tert-butyl ester 2 that was further reacted with the mal-
eimido peptide EMC-D-Ala-Phe-Lys(Boc)-Lys-OH. The MTX
prodrug 3 was obtained after removal of the protective
groups, chromatography on RP silica gel and lyophiliza-
tion. Compound 3 displays an excellent water-solubility
(A30 mg/mL in water) and is conveniently administered
in buffer solution.
In order to show that prodrug 3 binds rapidly and
selectively to endogenous HSA, HPLC-studies with human
plasma were performed at k = 300 nm (see Fig. 1). A chro-
matogram of pure human plasma recorded at this wave-
length shows two main signals: a sharp peak eluting at
4 min as well as a smaller broad peak at l32 min, the lat-
ter can be assigned to HSA. Figure 1 depicts the chroma-
tographic profile on a reversed-phase system of 3 incu-
bated with human blood plasma for 2 min. While the
unbound prodrug elutes at l27 min, this signal disap-
pears almost completely after incubation with human
plasma, and a single peak eluting at the retention time of
HSA (l32 min) is observed. In our previous work on albu-
min-binding prodrugs with doxorubicin, camptothecin,
and platinum derivatives, we have shown that this fast
reaction results from a Michael addition between the
maleimide group and the sulfhydryl group of the cys-
teine-34 position of serum albumin [10, 11, 18–22].
The plasma stability of the albumin conjugate of 3 was
evaluated by incubation of 3 with human blood plasma
and subsequently analyzing samples after 2 min and
24 h through HPLC. From the decrease of peak area the
loss of MTX after 24 h was determined to be a10%.
Results and discussion
Results
We designed a prodrug of MTX that consists of the drug,
a lysine spacer, and an enzymatically cleavable peptide
linker bearing the albumin-binding maleimide group.
The MTX's glutamic acid moiety possesses two carboxy-
late groups that can serve as functional groups for attach-
ing the drug to carrier molecules. However, it has been
shown that a free a-carboxylate group is essential for
retaining the drug's efficacy whereas chemical modifica-
tions of the c-carboxylate group are much better toler-
ated [17]. Therefore, our synthesis of the prodrug started
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Arch. Pharm. Chem. Life Sci. 2007, 340, 389–395
Albumin-Binding Prodrug of Methotrexate
391
Scheme 1. Synthesis route of 3.
Figure 3. Chromatograms of incubation studies with HSA-3 in
the presence of cathepsin B at pH 5.0 and 378C after 0 h, 1 h,
4 h, and 24 h.
Figure 2. Chromatograms of incubation studies with HSA-3 in
the presence of plasmin at pH 7.4 and 378C after 0 h, 1 h, 4 h,
and 20 h.
For assessing the cleavage properties of the albumin- son with a reference substance, the structure of the liber-
bound form of 3, the HSA conjugate (HSA-3) of the pro- ated MTX derivative was confirmed to be H-Lys(c-MTX)-
drug was prepared by incubating with an excess of 3 with OH which results from a proteolytic cleavage of the Lys-
HSA. A solution of HSA-3 was then incubated with either Lys bond.
human plasmin or cathepsin B, and samples were ana-
In order to show that cleavage of the macromolecular
lyzed by HPLC over a period of 20–24 h. In both cases, a prodrugs also occurs in the presence of native tumor tis-
time-dependent cleavage of the D-Ala-Phe-Lys linker was sue, HSA-3 was incubated with homogenized OVCAR-3
observed. The chromatograms in Figs. 2 and 3 illustrate material (ovarian carcinoma xenograft that was used in
the decrease in peak area of HSA-3 and the formation of a the in vivo experiment, see below Section 3.4). The sam-
single cleavage product eluting at l4 min. (Due to the ples were homogenized according to two different proto-
use of buffer containing D-cysteine in the experiments cols [23]: while homogenization at pH 5.0 should retain
with cathepsin B, an additional small peak of constant activity of lysosomal proteases, such as cathepsins, a
height eluting at 3.5 min can be observed.) By compari- work-up at pH 7.4 was performed to preserve the extracel-
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vious in vivo investigations which showed that 46100
mg/kg of MTX is the approximate MTD in nude mice [24].
The results of the xenograft experiment are summarized
in Fig. 5. Treatment with the prodrug 3 resulted in a clear
response (T/C max. 29%) whereas mice treated with
methotrexate showed no antitumor effects at all. Both
the prodrug and MTX were well-tolerated and no
decrease in body weight could be observed in either case.
Discussion
Coupling anticancer agents to macromolecular carriers
is a promising strategy for passively targeting solid
tumors. Among macromolecular prodrugs of the folic
acid antagonist MTX, only the albumin conjugate MTX-
HSA gained clinical importance and was assessed in vari-
ous clinical trials [25–27]. The conjugate is obtained
from directly coupling the drug to lysine residues of
human serum albumin and is preferably adjusted to a
loading rate of close to one equivalent MTX per molecule
of albumin [28, 29]. Since this condensing reaction is not
regioselective, MTX-HSA represents a complex mixture of
a- and c-amides with the drug bound to different lysine
positions of the protein. In addition, preactivation of
MTX with carbodiimides results in a considerable degree
of racemization producing a mix of D,L-MTX derivatives
[24]. Although there are some indications that MTX-HSA
acts as a prodrug by being lysosomally digested, the
release mechanism of the drug or respective drug-pep-
tide derivatives still remains unclear [30].
Figure 4. Chromatograms of incubation studies with HSA-3 in
the presence of OVCAR-3 homogenate at pH 5.0 at 378C after
4 h and at pH 7.4 at 378C after 24 h.
Our approach focused on designing a chemically well-
defined MTX-HSA conjugate 3 with distinct cleavage
properties. For this purpose, we chose the short peptide
sequence D-Ala-Phe-Lys (which is known to be efficiently
Figure 5. Curves depicting tumor growth inhibition of subcutane-
ously OVCAR-3 xenografts under therapy with methotrexate
and prodrug 3.
lular enzymes (including plasmin) in their active forms. cleaved at its C-terminus by the proteases plasmin and
As shown in Fig. 4, incubation with OVCAR-3 homoge- cathepsin B) as a crosslinker to link the drug with an
nate at pH 7.4 resulted in very little cleavage even after a albumin-binding maleimide group. This anchor ensures
24 h incubation whereas the conjugate was readily a rapid and regioselective coupling to the cysteine-34
cleaved in homogenate at pH 5.0 (approximately 90% position of HSA and thus can be successfully employed
after 4 h of incubation). In all cases, only one cleavage for the in situ generation of drug-albumin conjugates as
product (H-Lys(c-MTX)-OH) was observed (other visible demonstrated in our previous work [18]. Due to the fact
peaks are caused by the homogenized material). Also in that the peptide sequence must be linked to MTX via its
tumor homogenate, a further (enzymatic) degradation to C-terminus, we incorporated the amino acid lysine as a
free MTX was not detected.
diamine spacer. Upon cleavage of the Lys-Lys bond we
Subsequently, the in vivo antitumor efficacy of the MTX expected the MTX derivative H-Lys(c-MTX)-OH to be
prodrug 3 was evaluated in xenografted nude mice released as the active species. H-Lys(c-MTX)-OH in which
(human ovarian carcinoma OVCAR-3) in comparison to the e-amino group of lysine is bound to the c-position of
methotrexate. Compound 3 was assessed at a dose of the drug was shown to inhibit the dihydrofolate reduc-
4615 mg/kg (i.v.) MTX equivalents. This dose was chosen tase nearly as efficiently as the drug itself [31].
after an orientating toxicity study in healthy mice in
HPLC studies performed with 3 or the HSA conjugate
which a dose of 8615 mg/kg was well-tolerated produc- of 3 (HSA-3) confirmed the expected binding and cleavage
ing only a slight decrease in body weight (-5% from day 1 properties: (1) after incubation with human plasma, the
to day 28). The dose of MTX was chosen according to pre- major amount of 3 binds to HSA within 2 min; (2) the
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Arch. Pharm. Chem. Life Sci. 2007, 340, 389–395
Albumin-Binding Prodrug of Methotrexate
393
protein contained approximately 60% free thiol groups as
assessed with the Ellmann's test. The buffers used were vacuum-
filtered through a 0.2 lm membrane (Sartorius, Germany) and
thoroughly degassed with ultrasound prior to use. Enzymati-
cally active cathepsin B and plasmin were purchased from Cal-
biochem (Bad Soden, Germany). OVCAR-3 xenograft material
was gratefully received from Dr. I. Fichtner (EPO Exp. Pharmakol.
& Onkol. GmbH, Berlin, Germany). Lyophilization was perform-
ed with a lyophilizator Alpha 2-4 (Christ, Germany). ESI-MS spec-
tra were obtained on a Finnigan MAT 312 with associated MAT
SS 200 data system (Thermo Electron Corporation, Bremen, Ger-
many).
conjugate HSA-3 is sufficiently plasma-stable; (3) efficient
cleavage of the conjugate can be observed in the presence
of either human plasmin or cathepsin B; (4) cleavage
results in the formation of H-Lys(c-MTX)-OH as the only
low-molecular weight cleavage product. Since HSA-3 is
cleaved by either intra- or extracellular proteases, we also
expected a release of H-Lys(c-MTX)-OH in homogenized
native tumor material. For this purpose, we prepared two
samples of OVCAR-3 material at different pH. The first
one, worked up at pH 5, should preserve lysosomal pro-
teolytic activity whereas the second one, prepared at neu-
tral pH, was assumed to maintain extracellular enzyme
activity. Interestingly, when incubating HSA-3 with the
respected tumor homogenates, a clear difference in the
cleavage rate was observed. The conjugate was degraded
much more effectively in the homogenate at pH 5. At
pH 7.4 we expected to see plasmin activity but almost no
cleavage occurred. A reason for this can be that OVCAR-3
lacks a high plasmin expression, likewise it cannot be
excluded that plasmin activity was lost during the
homogenization process (e.g. by deactivation with a2-
antiplasmin or the degradation by other proteases).
The finding that H-Lys(c-MTX)-OH is not further
degraded to MTX and lysine is in accordance with pre-
vious work by Fitzpatrick and Garnett who showed that
H-Lys(c-MTX)-OH withstands exposure to harsh proteo-
lytic conditions as found in liver tritosomes [32]. How-
ever, the results of the in vivo experiment suggest that
the released MTX-lysine derivative is highly active per se.
Compared to methotrexate, 3 exhibited enhanced effi-
cacy at a much lower dose. Therefore, it seems that fur-
ther degradation to MTX is not necessary although it
would be interesting to compare 3 with an analogous
prodrug incorporating a more sophisticated (self-immo-
lative) spacer that enables the release of pure MTX.
HPLC method
HPLC for binding and cleavage studies with 3 and its albumin
conjugate HSA-3 was performed using a Gilson 321 pump (Gil-
son Middleton, MI, USA), a Pharmacia LKB Uvicord VW2251 UV
detector (k = 300 nm; Pharmacia-LKB, Uppsala, Sweden), and Kro-
maSystem Software (Northstar Scientific, UK); column: Waters
300 ꢀ (Waters Corporation, Milford, MA, USA), Symmetry C18
(4.66250 mm) with pre-column; chromatographic conditions:
flow: 1.2 mL/min, mobile phase A: 15% CH3CN, 85% 20 mM
potassium phosphate (pH 7.0), mobile phase B: 70% CH3CN, 30%
20 mM potassium phosphate (pH 7.0); gradient: 0–20 min 100%
mobile phase A; 20–45 min increase to 100% mobile phase B;
45–50 min 100% mobile phase B; 50–56 min decrease to 100%
mobile phase A; injection volume: 50 lL.
Chemistry
MTX-a-Ot-Bu-c-OMe (1)
To a suspension of 4-amino-4-deoxy-N10-methylpteroic acid
(APA) (3.15 g, 8.71 mmol, 90% purity) in 200 mL of anhydrous
DMF was added 6 mL of triethylamine and BOP (5.23 g,
11.8 mmol). After stirring for 30 min at room temperature, H-
Glu(OMe)-Ot-Bu.HCl (2.38 g, 9.40 mmol) was added, and stirring
was continued for 16 h. The dark-orange mixture was filtered
over celite, and the filtrate was concentrated in vacuo to a final
volume of 100 mL. Subsequently, the crude product was precipi-
tated by pouring the solution slowly into vigorously stirred
diethyl ether (2000 mL). The suspension was left at 48C for 3 h
after which precipitation was complete. The solids were col-
lected by filtration and washed twice with cold diethyl ether.
The crude product was recrystallized from 1000 mL of ethanol
to yield 4.08 g (89%) of crude diester 1 which was used in the
next step without further purification.
In summary, we developed the first albumin-binding
prodrug of methotrexate 3 that is cleaved by either intra-
or extracellular proteases and demonstrated superior in-
vivo efficacy over MTX thus warranting further preclini-
cal assessment.
MTX-a-Ot-Bu (2)
Compound 1 (4.05 g, 7.72 mmol) was suspended in 150 mL of
ethanol/water 1 : 1 in an unstoppered Erlenmeyer flask.
Ba(OH)268 H2O (2.02 g, 6.40 mmol) was added, and, after stir-
ring, the initially cloudy mixture cleared off within 10 min. Stir-
ring was continued at room temperature for at least 16 h, and
progress of the reaction was monitored using TLC (CHCl3/MeOH,
5 : 1, +1% AcOH). In some cases it was found to be necessary to
continue stirring until conversion was complete (up to three
days). Subsequently, the solution was filtered over celite, and a
saturated solution of Na2SO4 (equimolar to Ba(OH)2) was added.
The mixture was left at 48C for 5 h after which precipitation of
BaSO4 was complete, and was then filtered over celite. Remain-
ing ethanol was removed under reduced pressure, and the solu-
tion was washed with 3650 mL of chloroform. After acidifying
Experimental
Chemicals, materials, and spectroscopy
4-Amino-4-deoxy-N10-methylpteroic acid (APA) was a gift from
Klinge Pharma (Munich, Germany); EMC-D-Ala-Phe-Lys(Boc)-Lys-
OH was custom-made by JPT (Berlin, Germany); organic solvents:
HPLC grade (Merck, Darmstadt, Germany). All other chemicals
used were at least reagent grade and obtained from Sigma-
Aldrich, Fluka (Buchs, Switzerland), or Merck and used without
further purification. Human serum albumin (5% solution) was
purchased from Octapharma GmbH (Langenfeld, Germany). The
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the aqueous phase with 0.5 M HCl to pH 3, a yellow solid precipi-
tated which was filtered off, washed with water, 2-propanol,
diethyl ether, and pentane, and dried in high vacuum to afford
2.68 g (68%) of pure monoester 2. 1H-NMR (DMSO-d6): d = 1.38 (s,
9H, Ot-Bu), 1.82–1.96 (m, 2H, b-CH2), 2.03–2.22 (m, 2H, c-CH2),
3.18 (s, 3H, NCH3), 4.06 (m, 1H, a-CH), 4.76 (s, 2H, 9-CH2), 6.60 (bs,
2H, 4-NH2), 6.81 (d, 2H, J = 8.7 Hz, H-3‘, H-59), 7.40 (bs, 1H, 2-NH2),
7.68 (bs, 1H, 2-NH2), 7.75 (d, 2H, J = 8.7 Hz, H-29, H-69), 8.56 (s, 1H,
H-7), 10.10 (bs, 1H, NH). 13C-NMR (DMSO-d6): d = 26.51 (C-b), 27.63
(OC(CH3)3), 31.10 (C-c), 39.01 (C-11), 54.39 (C-a), 54.74 (C-9), 79.53
(OC(CH3)3), 111.00 (C-39, C-59), 121.31 (C-19), 121.50 (C-4a), 128.69
(C-29, C-6k), 145.88 (C-6), 149.02 (C-7), 150.63 (C-49), 155.08 (C-8a),
162.58 (C-4), 162.74 (C-2), 165.70 (C-79), 171.65 (COOt-Bu), 176.57
(COOH). MS (APCI) m/z = 525.4 [M+H]+, (100).
Incubation studies of HSA-3 with plasmin
100 lL of the HSA-3 stock solution (700 lM) were mixed with
500 lL of phosphate buffer (4 mM sodium phosphate, 150 mM
NaCl, pH 7.4) and 20 lL of a plasmin solution (1.87 mg/mL,
18.7 units/mL) and incubated at 378C. Samples were collected
after 2 min, 1 h, 4 h, and 20 h and were analyzed by HPLC.
Preparation of OVCAR-3 tissue homogenates
For preparing the tumor homogenates two protocols were used:
the first at pH 5.0 that primarily liberates lysosomal proteases,
the second at pH 7.4 that is used for extracting extracellular pro-
teases. All following steps were carried out on ice. Tissues of
OVCAR-3 xenografts were cut into small pieces, and 200 mg sam-
ples were transferred in a 2 mL-Eppendorf tube to which was
added 800 lL of buffer (50 mM Tris-HCl buffer, pH 7.4, contain-
ing 1 mM monothioglycerol or 50 mM sodium acetate buffer,
pH 5.0 containing 100 mM sodium chloride, 4 mM EDTA-Na2,
and 0.1% Brij 35). Homogenization was carried out with a micro-
dismemberator at 3000 rpm for 3 min with the aid of glass balls.
Subsequently, the samples were centrifuged at 5000 rpm for
10 min at 48C. The supernatant was aliquoted and kept frozen at
-788C prior to use.
EMC-D-Ala-Phe-Lys-Lys(c-MTX)-OH (3)
To a solution of 2 (125 mg, 244 lmol) in 0.7 mL DMF was added
HATU (93 mg, 244 lmol) and DIEA (186 lL, 1.10 mmol). The mix-
ture was stirred vigorously for 1 min and then transferred to a
solution of EMC-D-Ala-Phe-Lys(Boc)-Lys-OH N 2 TFA (200 mg,
222 lmol) and DIEA (186 lL, 1.10 mmol) in 3.3 mL DMF. After
1 h stirring at room temperature, the reaction mixture was
poured slowly into 300 mL of vigorously stirred diethyl ether.
The solids were collected by filtration, washed twice with diethyl
ether and dried in vacuo. Subsequently, both Boc and tert-butyl
protective groups were removed by treating the product with
4 mL of TFA/CH2Cl2 (1 : 1) over 1 h at room temperature. The
product was precipitated by pouring the solution slowly into vig-
orously stirred diethyl ether (300 mL), the solids were collected
by filtration, and purification of the crude product was carried
out by column chromatography (C18-RP silica gel, water/MeCN,
70 : 30, +0.1% TFA). After lyophilization, 148 mg (54%) of 3 were
obtained as a yellow solid. Purity determined by HPLC (k =
370 nm): A99%. MS (ESI) m/z = 1122.3 [M+H]+ (100), 1144.4
[M+Na]+ (73).
Incubation studies of HSA-3 with OVCAR-3 tissue
homogenates
100 lL of the HSA-3 stock solution (700 lM) were either mixed
with OVCAR-3 tumor homogenate at pH 7.4 or pH 5.0 and incu-
bated at 378C. Samples were collected over 24 h and were ana-
lyzed by HPLC.
In vivo testing
Orientating toxicity studies
The experiment was carried out using 6–8 weeks old female
DAB/1 mice (Charles River, Sulzfeld, Germany) that were treated
i.v. on days 1, 4, 8, 11, 14, 18, 22, and 25 with 3 at a dose of
15 mg/kg MTX equivalents freshly dissolved in glucose-phos-
phate buffer (10 mM sodium phosphate, 5% D-(+)-glucose, pH 6.4,
5 mg/mL). Animal weight, abnormal animal behavior, and
potential signs of sickness were documented. Experimental pro-
tocols had been approved by the Ethics Committee for Animal
Experimentation according to the United Kingdom Coordina-
tion Committee on Cancer Research Guidelines and the Ethics
Committee of the University Freiburg.
HSA conjugate of 3 (HSA-3)
To 5.16 mg of 3 were added 10.3 mL of HSA solution (Octa-
pharma, 5%). The mixture was shaken for 2 h at room tempera-
ture and concentrated with the aid of Centriprepsm to a final vol-
ume of approximately 3 mL. The concentration of HSA-3 was
determined photometrically at 370 nm (e370 = 7420 M– 1cm– 1) to
be 700 lM.
In-vitro testing
In vivo efficacy
For the in-vivo testing of 3 in comparison with MTX female NMRI
nu/nu mice (M&B A/S, Ry, Denmark) were used. The mice were
held in laminar flow shelves under sterile and standardized
environmental conditions (25 l 28C room temperature,
50 l 10% relative humidity, 12-hour light-dark-rhythm). They
received autoclaved food and bedding (ssniff, Soest, Germany)
and acidified (pH 4.0) drinking water ad libitum. All animal
experiments were performed under the auspices of the German
Animal Protection Law. A number of 107 cells of human ovarian
cancer cells OVCAR-3 from in vitro culture were transplanted
subcutaneously (s.c.) into the left flank region of anaesthetized
(40 mg/kg i.p.; Radenarkon, Asta Medica, Frankfurt, Germany)
mice on day zero. Mice were randomly distributed to the experi-
mental groups (seven mice per group). When the tumors were
grown to a palpable size, treatment was initiated (see Fig. 5).
Incubation studies of 3 with human blood plasma
Compound 3 (1.00 mg) were dissolved in 200 lL of sterile-filtered
glucose-phosphate buffer (10 mM sodium phosphate/5% D-glu-
cose buffer, pH 6.4). 20 lL of this solution were added to 380 lL
of human plasma and incubated at 378C. Samples were collected
after 2 min, 1 h, and 20 h and were analyzed by HPLC.
Incubation studies of HSA-3 with cathepsin B
180 lL of the HSA-3 stock solution (700 lM) were mixed with
270 lL of acetate buffer (50 mM sodium acetate, 100 mM NaCl,
4 mM EDTA-Na2, pH 5.0) containing L-cysteine (8 mM) and 90 lL
of a cathepsin B solution (71.7 lg/mL, 110 mU) and incubated at
378C. Samples were collected after 2 min, 1 h, 4 h, and 24 h and
were analyzed by HPLC.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 389–395
Albumin-Binding Prodrug of Methotrexate
395
Mice were treated intravenously at day 7, 14, 21, and 28 with
either glucose-phosphate buffer (10 mM sodium phosphate, 5%
D-(+)-glucose, pH 6.4, 5 mg/mL), MTX (100 mg/kg), or 3 (15 mg/kg
MTX equivalents) freshly dissolved in glucose-phosphate buffer.
The injection volume was 0.2 mL/20 g body weight.
Tumor size was measured twice weekly with a calliper-like
instrument in two dimensions. Individual tumor volumes (V)
were calculated by the formula V = (length + [width]2)/2 and
related to the values on the first day of treatment (relative tumor
volume, RTV). Statistical analysis was performed with the U-test
(Mann and Whitney) with p a 0.05. The body weight of mice was
determined every 3 to 4 days.
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