New angiopep-modified doxorubicin (ANG1007) and etoposide (ANG1009) chemotherapeutics with increased brain penetration

Article abstract of DOI:10.1021/jm9016637

This report describes the synthesis and preliminary biological characterization of 2 (ANG1007) and 3 (ANG1009), two new chemical entities under development for the treatment of primary and secondary brain cancers. 2 consists of three doxorubicin molecules conjugated to Angiopep-2, a 19-mer peptide that crosses the blood-brain barrier (BBB) by an LRP-1 receptor-mediated transcytosis mechanism. 3 has a similar structure, with the exception that three etoposide moieties are conjugated to Angiopep-2. Both agents killed cancer cell lines in vitro with similar IC₅₀ values and with apparently similar cytotoxic mechanisms as unconjugated doxorubicin and etoposide. 2 and 3 exhibited dramatically higher BBB influx rate constants than unconjugated doxorubicin and etoposide and pooled within brain parenchymal tissue. Passage through the BBB was similar in Mdr1a (-/-) and wild type mice. These results provide further evidence of the potential of this drug development platform in the isolation of novel therapeutics with increased brain penetration.

Full text of DOI:10.1021/jm9016637

2814 J. Med. Chem. 2010, 53, 2814–2824
DOI: 10.1021/jm9016637
New Angiopep-Modified Doxorubicin (ANG1007) and Etoposide (ANG1009) Chemotherapeutics With
Increased Brain Penetration
†
Christian Che, Gaoqiang Yang, Carine Thiot, Marie-Claude Lacoste, Jean-Christophe Currie, Michel Demeule,*
†
†
‡
,†
ꢀ †
Anthony Regina, Richard Beliveau, and Jean-Paul Castaigne
†
‡
†
ꢀ
ꢀ
†Angiochem, 201 President Kennedy Avenue (PK-R220), Montreal, Queꢀbec, Canada H2X 3Y7, and ‡Laboratoire de Meꢀdecine Moleꢀculaire,
Universiteꢀ du Queꢀbec aꢁ Montreꢀal, Montreꢀal, Queꢀbec, Canada
Received November 10, 2009
This report describes the synthesis and preliminary biological characterization of 2 (ANG1007) and 3
(ANG1009), two new chemical entities under development for the treatment of primary and secondary
brain cancers. 2 consists of three doxorubicin molecules conjugated to Angiopep-2, a 19-mer peptide
that crosses the blood-brain barrier (BBB) by an LRP-1 receptor-mediated transcytosis mechanism. 3
has a similar structure, with the exception that three etoposide moieties are conjugated to Angiopep-2.
Both agents killed cancer cell lines in vitro with similar IC50 values and with apparently similar cytotoxic
mechanisms as unconjugated doxorubicin and etoposide. 2 and 3 exhibited dramatically higher BBB
influx rate constants than unconjugated doxorubicin and etoposide and pooled within brain parenchy-
mal tissue. Passage through the BBB was similar in Mdr1a (-/-) and wild type mice. These results
provide further evidence of the potential of this drug development platform in the isolation of novel
therapeutics with increased brain penetration.
Introduction
both doxorubicin and etoposide into brain tissue is dramati-
cally inhibited by the BBB.13,14
Doxorubicin and etoposide exhibit excellent therapeutic
activity against a variety of solid tumors.1-4 Doxorubicin,
a cytotoxic anthracycline antibiotic that intercalates into
DNA, is thought to promote cytotoxicity by inhibiting
DNA and RNA polymerases and by interacting with topo-
isomerase II to form DNA-cleavable complexes.5 Etoposide,
a semisynthetic derivative of podophyllotoxin used for differ-
ent malignancies and as first line treatment in small cell lung
cancer,6 is thought to promote cytotoxicity by disrupting cell
cycle progression, presumably by promoting DNA strand
breaks in combination with DNA topoisomerase II.7
Chemotherapy for malignant brain tumors often has lim-
ited efficacy, largely due to restricted blood-brain barrier
(BBBa) permeability for chemotherapeutic drugs.8 Intercellu-
lar tight junctions between capillary endothelial cells, a con-
tinuous (i.e., nonfenestrated) capillary endothelium, multiple
intracellular efflux pumps with broad substrate specificity,
and an array of intracellular and extracellular degradative/
metabolic enzymes all combine to produce the restrictive
diffusion barrier characterizing the BBB.9-12 Penetration of
We have developed a novel drug-development technology
called the engineered peptide compound (EPiC) platform that
exploits the endogenous LRP-1 receptor-mediated transcyto-
sis system. This platform is based on a proprietary 19-amino-
acid sequence, called Angiopep-2, that crosses the BBB by an
LRP-1 mediated mechanism. 1 (ANG1005),13,15 a new che-
mical entity (NCE) under development for the treatment of
primary andsecondary brain cancers, isthe firstagenttoreach
clinical trials based on this platform.16 1 (Figure 1) is com-
posed of one Angiopep-2 peptide conjugated to three mole-
cules of paclitaxel, a broad-spectrum antitumor agent that
inhibits reorganization of microtubules during interphase and
mitosis. In mice, 1 inhibited growth of orthotopic human
glioblastoma (U87 MG) brain tumors more potently than
paclitaxel alone and significantly increased animal survival
rates.16 In phase I clinical trials in humans, 1 reached thera-
peutic concentrations in brain tumors and produced signifi-
cant antitumor responses in patients with primary gliomas or
secondary brain metastases who had failed prior standard
therapy.17,18 The present study describes the synthesis and
preliminary biological characterization of two new Angiopep-
modified doxorubicin and etoposide derivatives with in-
creased brain penetration.
*To whom correspondence should be addressed. Phone: 514-987-
3000 ext. 4087/6697. Fax: 514-987-0246; E-mail: mdemeule@
angiochem.com.
a Abbreviations: LRP1, low-density lipoprotein receptor-related pro-
tein; pAb, polyclonal antibody; PBS, phosphate-buffered saline; BBB,
blood-brain barrier; DMSO, dimethylsulfoxide; UPLC, ultra perfor-
mance liquid chromatography; FA, formic acid; DIEA, N,N-diisopro-
pylethylamine; FmocOSu, N-(9-fluorenylmethoxycarbonyloxy) succini-
mide; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid;
DCM, dichloromethane; TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium tetrafluoroborate; RPC, reverse phase chromato-
graphy; DMAP, 4-dimethylaminopyridine.
Chemistry
Synthesis. The first derivative 2 consists of three molecules
of doxorubicin conjugated via a cleavable ester bond to one
Angiopep-2 peptide (Figure 1). For its synthesis (Scheme 1),
the primary amine in the sugar of doxorubicin was first
protected by an Fmoc group to provide intermediate 4 in
r
pubs.acs.org/jmc
Published on Web 03/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2815
Figure 1. Chemical structure of 1, 2, and 3. 1 , 2, and 3 are each composed of three molecules of paclitaxel, doxorubicin, and etoposide,
respectively, linked by cleavable ester to the Angiopep-2 peptide.
Scheme 1. Synthesis of 2
Scheme 2. Synthesis of 3

2816 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cheꢀ et al.
good yield (80%), which was then reacted with succinic
anhydride to afford, primarily, the key acid 5. Some bis-
acylated side-products, via the two hydroxyls, were also
formed (<15%) and removed by SiO2 purification. The free
acid 5 was activated by TBTU prior to in situ conjugation
with Angiopep-2. Under standard Fmoc deprotection con-
ditions, the desired product 2 was obtained in 17% yield after
purification on an AKTAexplorer system (GE Healthcare
of the HPLC-purified final products, as determined by mass
spectrometry, were consistent in each case with three drug
moieties conjugated to a single Angiopep-2 peptide. Thus,
the observed mass of purified 2 (LC-HRMS, ESI, m/z
2089.9674 [M þ 2], 1393.2419 [M þ 3], and 1045.4395
[M þ 4]) was consistent with the calculated mass of C197H242
-
N32O70 (4177.6428) (Figure 3A). Similarly, the observed
mass of purified 3 (m/z 2305.9327 [M þ 2], 1537.6443 [M þ 3],
1153.7463 [M þ 4], and 922.7970 [M þ 5]) was consistent with
the calculated mass of C218H281N32O79 (4610.8955) (Figure 3B).
2 and 3 with purities >95% were used in all subsequent
biological assays.
ꢀ
Life Sciences, Baie d’Urfe, Quebec, Canada; see Experimen-
tal Section).
The second derivative 3 consists of three molecules of etopo-
side conjugated via a cleavable ester bond to the Angiopep-2
peptide (Figure 1). For the synthesis of 3(Scheme 2), adimethyl
amino acetyl group was first introduced by acetylation at the 40
position of the phenol of etoposide to generate intermediate 6in
67% yield, which was then reacted with glutaric anhydride in
the presence of DMAP to produce, primarily, semiglutarate 7
at the 200 position in 40% yield. Some regioisomer 8 at the 300
position was also isolated in 5% yield. The assignment of
regioisomers 7 and 8 was performed by proton chemical
shifts (Table 1)19 and confirmed by correlation spectroscopy
(COSY) (data not shown). Regioisomer 7 was conjugated in
situ with Angiopep-2 as previously described to provide 3 in
26% yield.
3 was soluble in dextrose 5% in water (D5W) up to 20 mg/
mL. Similarly, the HCl salt of 2 could be dissolved at the
same concentration in D5W. The high solubility of 2 and 3
facilitated their in vitro and in vivo studies.
Analysis of End-Products. HPLC profiles of the final
synthetic products exhibited single peaks with retention
times for 2 (4.8 min) and 3 (5.4 min) that were distinct from
unconjugated Angiopep-2 (4.2 min) (Figure 2). The masses
Results and Discussion
In Vitro Cytotoxicity. The in vitro cytotoxic activities of 2
and 3 were evaluated using a thymidine incorporation assay.
To do so, human tumor cell lines were incubated for 48 h in
the presence of increasing concentrations of 2, 3, doxorubi-
cin, or etoposide. After aspirating off the old media, cells
were pulse-labeled for 2 h in fresh media containing [3H]-
thymidine. Uptake of tritium was evaluated in a β counter,
and the drug concentrations required to inhibit cell prolif-
eration by 50% (IC50) were calculated. Results from these
experiments showed that, in general, 2 and 3 had cytotoxic
activities that were comparable to their parental drugs
(doxorubicin and etoposide) in U87 MG glioblastoma,
SK-Hep-1 hepatocarcinoma, and NCI-H460 lung carcino-
ma cell lines (Table 2), albeit 3 appeared to be somewhat less
potent than etoposide in glioblastoma and lung carcinoma
cells.
For 2 and 3, as well as the earlier agent 1, conjugation of
the individual anticancer drugs to Angiopep-2 was achieved
via a cleavable ester bond. When doxorubicin was conju-
gated to Angiopep-2 via a noncleavable linker, however, the
Angiopep-2-doxorubicin conjugate lost its antiproliferative
activity, indicating that doxorubicin needed to be released
from the peptide to be active in cancer cells (data not shown).
To evaluate their in vitro half-lives, a release study was
performed on 2 and 3 by incubating the drugs in human
serum for 0-4 h and quantifying the release of doxorubicin
Table 1. Proton Chemical Shifts at the 200 and 300 Positions for
Etoposide, Compounds 6, 7, and 8
δ (ppm) in CD3OD
proton
etoposide19
6
7
8
200
300
3.25
3.53
3.32
3.52
4.74
3.78
3.44
4.76
Figure 2. HPLC analysis of Angiopep-2, 2, and 3. Representative chromatograms obtained for Angiopep-2, 2, and 3 showing three distinct
retention times.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2817
Figure 3. LC-MS analysis of 2, and 3. (A) Representative mass spectrum of 2 with monitoring at m/z = 4178. (B) Representative mass
spectrum of 3 with monitoring at m/z = 4610.
or etoposide by HPLC or LC/MS analysis. On the basis of
these results, the estimated half-lives for 2 and 3 were 24 and
63 min, respectively.
mately in a cell-cycle pause in G2. The role of cellular
membrane binding in this process remains unclear. Etoposide
is thought to produce breaks by either interacting directly
with topoisomerase II or by participating in the formation of
free radicals, which again pauses cells in G2. To analyze
whether 2 and 3 had similar cell cycle effects, U87 MG
glioblastoma cells were treated with 2 (33 nM), doxorubicin
(100 nM), 3 (1 μM), or etoposide (3 μM) for 48 h and cellular
DNA content was determined by flow cytometry (Table 3).
In the experiments comparing doxorubicin to 2, cells prior
to drug addition were distributed across the cell cycle in a
Cell Cycle Effects. Doxorubicin and etoposide arrest cells
in the G2 phase of the cell cycle,20,21 although the mechanism
may be different for both agents.22,23 The cytotoxic effect of
doxorubicin on malignant cells is thought to be related to
nucleotide base intercalation and cell membrane lipid bind-
ing. DNA intercalation of doxorubicin inhibits the action of
DNA and RNA polymerases and interacts with topoisome-
rase II to form DNA-cleavable complexes, resulting ulti-

2818 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cheꢀ et al.
Table 2. In Vitro Cytotoxicity of 2 and 3a
IC50 (nM)
glioblastoma
(U87 MG)
hepatocarcinoma
(SK-Hep-1)
lung carcinoma
(NCI-H460)
2
6.0
18
4.6
10
48
62
7.3
11
doxorubicin
3
etoposide
330
145
148
90
a Values are means of 3-5 experiments (n = 8).
Table 3. Effects of 2 and 3 on the Cell Cyclea
cell cycle phase (%)
molecules
G0/G1
S
G2/M
control
doxorubicin
2
58.2
30.8
32.3
56.8
8.9
14.1
27
8.9
10.1
5.1
59.9
57.3
38
control
etoposide
3
4.2
3.3
87
70.8
25.7
a U87 MG glioblastoma cells were treated for 24 h with no drug
(control), doxorubicin, 100 nM, or product 2, 33 nM. U87 glioblastoma
cells were also treated for 24 h with no drug (control), etoposide, 3 μM,
and product 3, 1 μM. DNA cellular content was analyzed by flow
cytometry. Results are presented as the percentage of cells in each cell
cycle phase. One representative experiment of three is shown.
Figure 4. In vivo brain uptake of 2. (A) Time course of brain uptake
of [125I]-2 (filled circles) and [14C]-doxorubicin (open circles) mea-
sured by in situ brain perfusion. Results were expressed as the
apparent volume of distribution (Vd) for the radiolabeled 2 or
doxorubicin in total brain homogenate. Lines represent best fits
to the data by least-squares regression. (B) After a 2 min perfusion
of [125I]-2 (filled bars) and [14C]-doxorubicin (open bars), brain
capillary depletion was performed and radioactivity was quantified
in total brain homogenate, brain capillary fractions, and brain
parenchymal fractions (Experimental Section). Results were ex-
pressed as the apparent volume of distribution (Vd) for the radio-
labeled drugs found in these brain compartments. All data represent
mean ( SD (n = 3-6 per time point).
normal logarithmic growth pattern, with 27% of the cells in
the G2/M phase of the cell cycle (Table 3). Addition of
doxorubicin increased the proportion of cells in G2/M to
59.9%, as expected from earlier published reports. Addition
of the Angiopep-2-doxorubicin derivative 2 also increased
the percentage of cells in G2/M to nearly the same extent,
57.3%, as unconjugated doxorubicin. Similarly, the propor-
tion of cells in G2/M rose from 38% in drug-naıve cells to
¨
87% and 70.8% after treatment with etoposide and the
Angiopep-2-etoposide derivative 3, respectively. Thus, treat-
ment with doxorubicin and 2, as well as etoposide and 3,
produced very similar cell cycle delays in G2/M, consistent
with the conjugated and unconjugated drugs having similar
molecular effects.
tent with an enhanced ability to cross the BBB. The observed
Kin values for 2 and 3 were similar to 1 (33 ( 0.2 ꢀ 10-4 mL/
g/s) (Table 3), suggesting similar influx kinetics for the three
drugs. In control experiments, [14C]-inulin, which is nor-
mally excluded from brain tissue in vivo, was perfused in the
presence of unlabeled 2 and 3 to verify the physical integrity
of the BBB (data not shown).
Blood-Brain Barrier Influx Rate Constants. We used an in
situ brain perfusion method,24,25 previously adapted in our
laboratory,15 to measure the transport of 2 and 3 into brain
tissue in mice (Experimental Section). In brief, [125I]-2, [14C]-
doxorubicin, [125I]-3, or [3H]-etoposide were perfused in situ
into the carotid artery for 0.5, 1, 2, or 4 min, followed by a
tracer-free washout period of 60 s. After perfusion, the
animals were sacrificed, the brain was surgically removed
and homogenized, and radioactivity in total brain tissue was
quantified. In the total brain homogenate, Vd increased in a
linear fashion for 2 (Figure 4A) and 3 (Figure 5A), exhibiting
no evidence of saturation after 4 min of perfusion for either
agent. By comparison, the Vd values for doxorubicin and
etoposide were low and remained relatively constant over the
same period, indicating little uptake of unconjugated drugs
into total brain tissue. The slopes of the curves, which
correspond to the BBB influx rate constants (Kin), are
presented in Table 4. The Kin for 2 (31 ( 0.7 ꢀ 10-4 mL/g/
s) was 12.4-fold greater than unconjugated doxorubicin
(2.5 ( 0.1ꢀ10-4 mL/g/s), while the Kin for 3 (22 ( 0.2 ꢀ
10-4 mL/g/s) was 24.4-fold greater than unconjugated eto-
poside (0.9 ( 0.1 ꢀ 10-4 mL/g/s). Thus, both new Angiopep-
2 drug conjugates exhibited dramatically higher BBB influx
rate constants than their unconjugated precursors, consis-
Parenchymal Uptake. To formally analyze whether 2 and 3
enter brain parenchyma or remain associated with the brain
capillary endothelium, we estimated the apparent volume of
distribution (Vd) for the two agents in total brain tissue,
brain capillaries, and brain parenchyma after in situ per-
fusion and brain capillary depletion. For the brain capillary
depletion, the brain was collected and homogenized as
described above and then subjected to differential centrifu-
gation through 35% Dextran 70.15 The radioactivity was
then quantified in the capillary (pellet) and parenchymal
(supernatant) fractions. At the 2 min time point, both 2
(Figure 4B) and 3 (Figure 5B) were present in parenchymal
tissue at levels significantly higher than unconjugated doxo-
rubicin and etoposide, indicating a higher rate of passage
across the BBB for both agents relative to their parental
precursors. By comparison, the Vd values for doxorubicin
and etoposide were low and remained relatively constant
over the same period, indicating little uptake of parental drugs
into parenchymal tissue or total brain tissue. Significant

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2819
Figure 5. In vivo brain uptake of 3. (A) Time course of brain uptake
of [125I]-3 (filled circles) and [3H]-etoposide (open circles) measured
by in situ brain perfusion. Results were expressed as the apparent
volume of distribution (Vd) for the radiolabeled 3 or etoposide in
total brain homogenate. Lines represent best fits to the data by least-
squares regression. (B) After a 2 min perfusion of [125I]-3 (filled bars)
and [3H]-etoposide (open bars), brain capillary depletion was
performed and radioactivity was quantified in total brain homo-
genate, brain capillary fractions, and brain parenchymal fractions
(Experimental Section). Results were expressed as the apparent
volume of distribution (Vd) for the radiolabeled drugs found in
these brain compartments. All data represent mean ( SD (n = 3-6
per time point).
Figure 6. In situ brain perfusion in wild-type and Mdr-1a (-/-)
knockout mice. Brain parenchyma uptake of (A) [125I]-2 and
[
14C]-doxorubicin (B) [125I]-3 and [3H]-etoposide measured by in
situ brain perfusion in wild-type (open bars) and Mdr1a (-/-) mice
(closed bars). Mice were perfused with 250 nM of [125I]-2,
750 nM [14C]-doxorubicin, 250 nM of [125I]-3, or 250 nM [3H]-
etoposide for 2 min prior to brain capillary depletion and quanti-
fication of radioactivity (Experimental Section). Results are ex-
pressed as the apparent volume of distribution (Vd) for the
radiolabeled drugs found in the brain parenchyma. Data represent
the means ( SD obtained for at least three mice. *P = 0.023, Mdr1a
(-/-) mice vs wild type in the doxorubicin-treated animals.
Table 4. Blood-Brain Barrier Influx Rate Constants for New Angio-
pep-2 Modified Anticancer Drugs and Their Respective Unmodified
Analogues
In mice, ABCB1 is encoded by three Mdr genes,32-35 and
previous studies have demonstrated that the Mdr1a isoform is
expressed at the BBB in mice.36
brain Kin (ꢀ 10-4 mL/g/s)
2
doxorubicin
31 ( 0.7
2.5 ( 0.1
22 ( 0.2
0.9 ( 0.1
33 ( 0.2
6.9 ( 0.5
The role of ABCB1 in the BBB can be conveniently
assayed using Mdr1a (-/-) mice, which lack this transpor-
ter and consequently exhibit greater brain accumulation of
many peripherally administered drugs.36-38 Consistent
with the demonstrated role of this pump in the efflux of
doxorubicin and etoposide at the BBB, mice bearing homo-
zygous mutations of Mdr1a exhibited >1.3- and 2-fold
increased brain penetration of unconjugated doxorubicin
(Figure 6A) and etoposide (Figure 6B) relative to wild type
control mice, respectively. These increases in doxorubicin
and etoposide brain uptake are similar to previous pub-
lished data.25 By comparison, brain parenchymal uptake
was similar in the wild type and mutant mice for both 2 and
3, indicating that they are not substrates for ABCB1 at the
BBB. Furthermore, the brain penetration of both 2 and 3
was much higher in the Mdr1a (-/-) knockout mice than
their parental drugs, suggesting that other efflux pumps
could limit the entry of the unconjugated drugs into brain
tissue. One candidate could be the breast cancer resistance
3
etoposide
1
pacitaxel
levels of 2 and 3 were also observed in the capillary fraction,
as expected from the extraordinarily high density of brain
capillaries.26
Transport of 2 and 3 in Mdr1a (-/-) mice. Brain capillary
endothelial cells contain multiple efflux pumps that protect the
brain from unwanted chemical assaults and contribute to the
intrinsic or acquired multidrug resistance (MDR) that can arise
after prolonged chemotherapy.27-29 In humans, one of the
most important efflux pumps expressed at the BBB is the ATP-
binding cassette (ABC) transporter protein B1 (ABCB1) also
known as MDR1 or P-glycoprotein (P-gp). At the BBB, this
efflux pump limits the entry to the brain of a vast array of
anticancer agents, including doxorubicin and etoposide.30,31

2820 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cheꢀ et al.
The results presented here and in previous publications13,15,16,41
lay the groundwork for assessing the potential of the EPiC
platform in neurotherapeutic development. Several key fea-
tures of the technology have now been documented. First,
NCEs developed to date using Angiopep-2 have all exhibited
highly efficient transport across the BBB. Thus 2, 3, and 1
each had significantly higher BBB Kin values than doxoru-
bicin, etoposide, and paclitaxel, respectively. Moreover, the
Kin values for all three agents compared favorably to glucose
(63 ꢀ 10-4 mL/g/s in rats),42 which crosses the BBB by a
specific carrier-mediated mechanism; morphine (1.6 ꢀ 10-4
mL/g/s in rats),43 which crosses the BBB by nonspecific
diffusion; and receptor-associated protein (1.0 ꢀ 10-4 mL/
g/s),44 which crosses the BBB by LRP-1 receptor-mediated
transcytosis. Moreover, it is noteworthy that 2 and 3 both
penetrated well into tumor tissue, encouraging further devel-
opment of these agents as therapies for brain cancers.
Second, the tolerance of the Angiopep-2 peptide to che-
mical modification appears to be flexible. Angiopep-2 is a 19-
amino-acid sequence derived from the Kunitz-type domain
present on some LRP-1 ligands, such as aprotinin, bikunin,
amyloid precursor protein, and tissue factor pathway inhi-
bitor.15 We now have evidence that 2, 3, and 1, each of which
carries three separate drug moieties, cross the BBB effi-
ciently. The molecular mechanism explaining how these
modifications to Angiopep-2 affect or modulate LRP-1
binding remains to be established. It should also be noted
that doxorubicin, etoposide, and paclitaxel, although differ-
ent in their chemical properties and hydrophilicities, are
nonetheless relatively small. Currently, modification of
Angiopep-2 with larger biologicals, including therapeutic
peptides, monoclonal antibodies, and siRNA, is under in-
vestigation in our laboratory.45
Finally, no evidence to date would suggest that conjuga-
tion of the Angiopep-2 sequence to anticancer agents dra-
matically inhibits their activities. In this and earlier16 studies,
the in vitro cytotoxic activities of 2, 3, and 1 were similar to
those of their unconjugated precursors, doxorubicin, etopo-
side, and paclitaxel, respectively. Moreover, 1 is currently in
phase I clinical trials and has shown promising activity
against primary and secondary brain cancers.17,18 The prior
data are consistent with our recent release studies, which
indicate that the anticancer moieties are likely cleaved in vivo
from Angiopep-2 conjugates prior to performing their anti-
proliferative activities. Again, the generality of these conclu-
sions will require an analysis of more agents, including larger
biologics, but preliminary evidence indicates that function-
ality may also remain after conjugation of neuroactive
peptides, antibodies, and siRNAs to Angiopep-2.45
Figure 7. Tissue distribution in mouse brain with U87 glioma
tumors. Brain biodistribution after intravenous bolus injection of
(A) [125I]-2 (filled bars) and [14C]-doxorubicin (open bars) and (B)
[
125I]-3 (filled bars) and [3H]-etoposide (open bars) measured in
normal brain and brain tumors. Mice were injected with 15 mg/kg of
125I]-2, 6 mg/kg [14C]-doxorubicin, 20 mg/kg of [125I]-3, or 8 mg/kg
[
[3H]-etoposide. Thirty min after injection, mice were perfused by the
heart, brain tissues were dissected and weighed, and radioactivity in
the brain tumor (implanted hemisphere) and normal brain tissue
(contralateral hemisphere) was quantified (Experimental Section).
Results are expressed as the % of injected dose per gram of tissue.
Data represent the means ( SD obtained for three mice (*P < 0.05,
** P < 0.01).
protein (BCRP or ABCG2), which is also expressed at the
BBB in rodents.14 Both doxorubicin and etoposide have
been reported to be substrates for BCRP.39,40 Overall, the
preceding results indicate that drugs and other substrates of
the ABCB1 efflux pump at the BBB may be modified with
Angiopep-2 to produce derivatives with enhanced brain
uptake.
Increased Brain Tissue Distribution. Finally, we measured
the accumulation of 2 and 3 in an orthotopic mouse brain
tumor model (Figure 7). Substantial differences were ob-
served in the biodistribution patterns of the two conjugates
compared to their parental drugs (doxorubicin and etopo-
side). For both 2 and 3, penetration into normal brain tissue
and tumor tissue after an intravenous bolus injection was
significantly higher than the parental drugs. When the
percentage of injected dose of drug per gram of tissue was
calculated, penetration of 2 increased by 3-fold in normal
brain tissue and by 2-fold in brain tumor tissue compared to
doxorubicin. By comparison, penetration of 3 increased by
15-fold in normal brain tissue and by 13-fold in tumor tissue
compared to etoposide.
Conclusions
The identification of new neurotherapeutics that can cross
the BBB has proven difficult in the past, and novel methodo-
logies for enhancing cross-BBB transport have long been
needed. The EPiC platform is based on a 19-amino-
acid peptide (Angiopep-2) that shows high transport across
brain capillary endothelial cells via an LRP1-mediated me-
chanism.13,15,16,41 Using Angiopep-2, new chemical entities
were created with enhanced transport across the BBB. The
first agent developed with this platform was 1 , which carries
three paclitaxel molecules conjugated to Angiopep-2. This
study examined two new agents, 2 and 3, which carry three
molecules of doxorubicin and etoposide, respectively, conjugated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2821
toAngiopep-2. Bothagents killed cancer celllines in vitro with
similar IC50 values and with apparently similar cytotoxic
mechanisms as unconjugated doxorubicin and etoposide. 2
and 3 also exhibited dramatically higher BBB influx rate
constants than unconjugated doxorubicin and etoposide
and pooled within brain parenchymal and tumor tissue.
Passage through the BBB for both agents was not increased
in mice lacking the ABCB1 efflux pump, implying their
transport bypasses this pump system, which distinguishes
the new agents from unconjugated doxorubicin and etopo-
side. In total, these results provide evidence that the novel
drug development platform described here can be used
broadly to isolate a range of small-molecule neurotherapeu-
tics with increased brain penetration.
127.61, 125.58, 121.11, 120.50, 120.38, 119.02, 112.08, 111.76,
77.0, 69.85, 67.18, 66.14, 62.64, 57.25, 47.48, 37.11, 36.15, 34.41,
32.07, 17.36. LC-HRMS (ESI, MicrOTOF), m/z calcd for
C42H39NO13 765.2421, found 788.2425 (M þ Na).
N-Fmoc Doxorubicin Hemisuccinate (5). DIEA (0.17 mL,
1.0 mmol) was added dropwise to a solution of N-Fmoc doxo-
rubicin 4 (0.28 g, 0.366 mmol) and succinic anhydride (0.11 g, 1.1
mmol) in DMF (20 mL) under stirring. The mixture was stirred
at room temperature and monitored by UPLC. After two days,
the reaction did not progress any more. The solvent was
removed, and the resulting residue was purified using Silicycle
siliaflash 40 g cartridge on Biotage system (2% to 15% MeOH in
DCM) to give 5 as a red powder 100 mg, 33%. UPLC purity,
95%. 1H NMR (600 MHz, CDCl3) δ (ppm) 7.96 (m, 1H), 7.74
(m, 2H), 7.56 (m, 2H), 7.36 (m, 2H), 7.29 (m, 2H), 7.06 (m, 2H),
5.45 (m, 1H), 5.35 (m, 2H), 5.2 (m, 2H), 4.35 (m, 1H), 4.18
(m, 2H), 4.05 (s, 3H), 3.8 (m, 2H), 3.6 (m, 1H), 3.23
(m, 1H), 2.95 (m, 1H), 2.79 (t, 2H, J = 7.0 Hz), 2.71 (t, 2H,
J = 7.0 Hz), 1.98-2.2 (m, 4H), 1.23 (d, J = 6.3 Hz, 3H). 13C
NMR (150 MHz, CDCl3) δ (ppm) 207.75, 187.63, 187.15,
175.07, 172.59, 172.49, 161.55, 156.34, 155.56, 144.42, 141.88,
136.40, 135.65, 133.97, 128.24, 128.23, 127.60, 125.60, 121.23,
120.49, 120.36, 118.99, 112.13, 111.78, 101.18, 98.54, 70.32,
69.99, 67.93, 66.95, 66.32, 62.48, 57.21, 47.81, 36.0, 33.94,
30.89, 30.66, 29.46, 29.35, 29.27, 17.26. LC-HRMS (ESI, Mi-
crOTOF), m/z calcd for C46H43NO16 865.2582, found 888.2710
(M þ Na).
Experimental Section
Reagents. Doxorubicin hydrochloride and etoposide were
purchased from Enzo Life Sciences (Plymouth Meeting, PA).
All other reagents and anhydrous solvents were purchased from
Sigma-Aldrich (St. Louis, MO) and used as received. NMR (1H,
13C) spectra were recorded on Varian AS600 spectrometers
(Palo Alto, CA) in CDCl3, CD3OD, or DMSO with solvent
resonance as the internal standard. Low- and high-resolution
mass spectra were recorded on Bruker micrOTOF spectro-
meters (Billerica, MA) using electron spray ionization (ESI-
TOF). The purity of the conjugate target compounds was
determined to be >95% by UPLC/MS on a Waters Acquity
UPLC spectrometer (Milford MA) and by HPLC on a Shimad-
zu SCL-10A HPLC (Columbia, MD). UPLC was conducted on
an Acquity UPLC BEH phenyl 1.7 μm column (2.1 mm ꢀ 50
mm) using a gradient of 10-90% MeCN-water (0.1% FA) at
0.5 mL/min. HPLC was conducted on a Taxsil column 3 μm (4.6
mm ꢀ 50 mm) using a gradient of 10-70% MeCN-water
(0.05% TFA) at 1 mL/min. Analytical thin-layer chromato-
graphy was performed on Merck 60F 254 precoated silica gel
plates. Flash column chromatography was performed on a
Biotage system (Charlottesville, VA) using Silicycle siliaflash
cartridges (230-400 mesh). Purifications were performed with a
30 RPC column on an AKTAexplorer 100 instrument (GE
Synthesis of 2. DIEA (0.25 mL, 1.44 mmol) was added
dropwise to a solution of 5 (599 mg, 0.692 mmol) and TBTU
(231 mg, 0.72 mmol) in DMF (21 mL) under stirring. The
mixture was stirred at room temperature for 50 min, thus a
solution of Angiopep-2 (671 mg, 0.229 mmol) in DMSO (2 mL)
and DMF (12 mL) was added. The mixture was stirred at room
temperature for 20 min. HPLC showed the reaction was com-
plete. After stirring for another 10 min, the solvent was removed.
The residue was purified using Biotage SNAP cartridge KP-
C18-HS 120 g (40% to 80% MeCN in water with 0.05% TFA)
to give Fmoc protected conjugate as a red powder 500 mg,
UPLC purity, 95%. To a solution of above Fmoc protected
conjugate (260 mg, 0.053 mmol) in DMSO (1 mL) and DMF
(12 mL) was added piperidine (20% in DMF, 1.5 mL). The
solution became blue. After stirring for 10 min, the solution was
cooled to 0 °C and treated with FA (0.5 M in DMF, 6 mL) to get
a clear red solution. The solvent was removed under reduced
pressure. The resulting residue was triturated with Et2O (3 ꢀ 10
mL) and EtOAc (3 ꢀ 10 mL). The resulting red solid was
purified using 30 RPC column on AKTAexplorer (10-40%
MeCN in water and 0.15% FA) to give 2 as a red powder 82 mg,
17% in two steps. UPLC purity, 95%. LC-HRMS (ESI, micrO-
TOF), m/z, calcd for C197H242N32O70 4177.6428, found
2089.9674 (2þ), 1393.2419 (3þ), 1045.4395 (4þ).
ꢀ
Healthcare, Baie d’Urfe, QC, Canada).
Animals. All animals were handled and maintained in accor-
dance with the Guidelines of the Canadian Council on Animal
Care.46 Animal protocols were approved by the Institutional
ꢀ
Animal Care and Use Committee of Universite du Quebec a
Montreal. Animals were obtained from Charles River Labora-
ꢀ
ꢁ
ꢀ
tories Inc. (Saint-Constant, QC, Canada) and were allowed to
acclimatize for 5 days before experiments. Brain perfusion
studies were performed on adult male Crl: CD-1 mice (25-
30 g, 6-8 weeks old) (Charles River Canada, St-Constant, QC,
Canada) and adult female CF-1 mice (Mdr1a (þ/þ) and (-/-),
30-40 g, 6-8 weeks old) (Charles River Inc., Wilmington, MA).
N-Fmoc Doxorubicin (4). DIEA (1.5 mL, 8.63 mmol) was
added dropwise to a solution of doxorubicin (2.0 g, 3.45 mmol)
and FmocOSu (2.32 g, 6.9 mmol) in DMF (35 mL) under
stirring. The mixture was stirred at room temperature for 2 h
and concentrated. The resulting residue was triturated with 0.1%
TFA in H2O (3 ꢀ 20 mL) and washed with Et2O (5 ꢀ 20 mL).
The resulting red solid was collected and repurified using a
Silicycle siliaflash 120 g cartridge on a Biotage system (2% to
10% MeOH in DCM) to give 4 as a red powder 2.1 g, 80%.
UPLC purity was 98%. 1H NMR (600 MHz, CDCl3) δ (ppm)
7.96 (m, 1H), 7.72 (m, 2H), 7.56 (m, 1H), 7.36 (m, 2H), 7.29 (m,
2H), 7.06 (m, 2H), 5.45 (m, 1H), 5.24 (m, 2H), 4.76 (m, 2H), 4.35
(m, 1H), 4.16 (m, 2H), 4.06 (s, 3H), 3.8 (m, 2H), 3.62 (m, 1H),
3.23 (d, 1H, J = 17.7 Hz), 2.35 (m, 1H), 2.16 (m, 1H), 1.8 (m,
1H), 1.31 (d, J = 6.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ
(ppm) 215.0, 187.53, 187.04, 163.16, 161.58, 156.48, 156.15,
155.85, 144.41, 141.85, 136.46, 135.95, 135.60, 133.95, 128.27,
Etoposide 40-Dimethylglycine (6). A mixture of etoposide
(235 mg, 0.4 mmol) and DMAP (73 mg, 0.6 mmol) in DMF
(4 mL) was stirred at room temperature for 20 min, then N,N-
dimethylacetyl chloride (96 mg, 0.52 mmol) was added in one
pot under stirring. After 30 min, the reaction was complete
according to HPLC. FA (1 M in DMF, 0.5 mL) was added, and
the solvent was concentrated to 1 mL. The resulting solution was
loaded to a 30 RPC column on AKTAexplorer for purification
(gradient 10-30% MeCN in H2O with 0.1% FA). After lyo-
philization, 6 (180 mg, 67%) was obtained as a colorless powder.
1H NMR (600 Hz, CD3OD) δ (ppm) 7.01 (1H, s), 6.56 (1H, s),
6.39 (2H, s), 5.98 (2H, d, J = 2.9 Hz), 5.05 (1H, d, J = 3.4 Hz),
4.77 (1H, q, J = 4.9 Hz), 4.68 (1H, d, J = 5.4 Hz), 4.66 (1H, d,
J = 7.8 Hz), 4.46 (2H, s), 4.45 (1H, dd, J = 10.3, 8.8 Hz), 4.31
(1H, t, J = 8.0 Hz), 4.17 (1H, dd, J = 10.3, 4.9 Hz), 3.68 (6H, s),
3.56 (1H, q, J = 10 Hz), 3.54 (1H, t, J = 9.3 Hz), 3.52 (1H, dd,
J = 14.2, 5.6 Hz), 3.32 (1H, m), 3.26 (1H, dd, J = 9.1, 4.1 Hz),
3.24 (1H, dd, J = 9.2, 5.4 Hz), 3.02 (6H, s), 2.96 (1H, m), 1.33
(3H, d, J = 4.9 Hz). 13C NMR (150 Hz, CD3OD) δ (ppm)

2822 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Cheꢀ et al.
175.26, 168.68, 151.35, 148.49, 147.01, 139.39, 132.74, 129.6,
127.28, 110.65, 110.45, 108.02, 102.19, 102.02, 99.25, 80.78,
75.06, 73.39, 72.41, 68.43, 68.01, 66.44, 59.73, 56.63, 56.47,
45.03, 43.86, 37.89, 20.99. LC-HRMS (ESI, micrOTOF) calcd
for C33H39NO14 673.2371, found 674.2534 (M þ 1).
PBS, pH 6.5. After incubation for 10 min at room temperature,
Iodo-beads were removed and the supernatants were applied to
a C18 column and purified by HPLC to remove free iodine.
After iodination, radiolabeled products were reanalyzed by
HPLC and results show that more than 95% of the radioactivity
was associated to 2 or 3.
In Vitro Cytotoxic Activity. For the thymidine uptake assay,
tumor cells were cultured in 96-well plates at a density of 5000
cells per well. After incubation of cells with anticancer drugs for
48 h, the medium was aspirated and cells were pulse labeled for
2 h at 37 °C with a medium containing 2 μCi/mL [methyl-3H]-
thymidine (GE Healthcare). Cells were harvested and placed in a
MicroBeta counter (1450 MicroBeta liquid scintillation and
luminescence Counter; Perkin-Elmer) for determination of tri-
tium uptake. Incorporated [3H]-thymidine was plotted as a
function of drug concentration. Concentration values required
to get a 50% inhibition (IC50) were estimated using GraphPad
software (La Jolla, CA).
In Vitro Half-Life Analysis. Stock solutions of 2 and 3 were
prepared at 1 mg/mL in D5W (dextrose 5% in water). 2 and 3 at
different concentrations (10-50 μg/mL) were incubated in hu-
man serum at 37 °C for 0 to 4 h. Serum proteins were pre-
cipitated by adding 370 μL of acetonitrile to 200 μL of serum,
samples were vortexed and centrifuged, and the acetonitrile
phases were transferred to clean glass tubes for LC/MS analysis.
In Vitro Cell Cycle Analysis Using Flow Cytometry. Expo-
nential U87 MG cells growing in 25 cm2 flasks were treated with
equimolar concentrations of 2 (33 nM), doxorubicin (100 nM), 3
(1 μM), or etoposide (3 μM) for 48 h. After treatment, attached
cells were released by trypsinization. Cells were centrifuged,
washed twice with PBS, and fixed with 70% ice-cold ethanol.
Fixed cells were resuspended in a DNA staining solution con-
taining 50 μg/mL propidium iodide and 0.5 mg/mL RNase in
10 mM Tris and 5 mM MgCl2. DNA cellular content was
analyzed by fluorescence-activated cell sorting (FACS) on a
FACS Calibur flow cytometer (Becton Dickinson, San Jose,
CA) using CellQuest Pro software, version 4.0.2.
Etoposide 40-Dimethylglycine 200-Hemiglutarate (7). A mix-
ture of 6 (655 mg, 0.97 mmol) and DMAP (18 mg, 0.15 mmol) in
chloroform (11 mL) was cooled to 0 °C. DMF (3 mL) and DIEA
(0.25 mL, 1.46 mmol) were added consecutively, followed by
glutaric anhydride (222 mg, 1.94 mmol). The reaction mixture
was stirred at room temperature, monitored by HPLC. After
2 days, the solvent was concentrated to 3 mL. The resulting
solution was loaded to a 30 RPC column on AKTAexplorer
for purification (gradient, 10-30% MeCN in H2O). After
lyophilization, 7 (305 mg, 40%) and 8 (38 mg, 5%) were
1
obtained as a white powder. 7, H NMR (600 Hz, CD3OD) δ
(ppm) 7.0 (1H, s), 6.53 (1H, s), 6.39 (2H, s), 5.99 (2H, d, J =
4.6 Hz), 4.97 (1H, q, J = 7.9 Hz), 4.94 (1H, d, J = 3.2 Hz), 4.78
(1H, q, J = 4.75 Hz), 4.74 (1H-200, dd, J = 9.2, 8.0 Hz), 4.68 (1H,
d, J = 5.6 Hz), 4.45 (2H, s), 4.41 (1H, dd, J = 9.6, 8.8 Hz), 4.29
(1H, t. J = 8.2 Hz), 4.15 (1H, dd, J = 10.0, 4.5 Hz), 3.78 (1H-300,
t, J = 9.4 Hz), 3.69 (6H, s), 3.61 (1H, t, J = 10.2 Hz), 3.42 (1H,
td, J = 9.6, 5.2 Hz), 3.33 (1H, dd, J = 8.7, 8.2 Hz), 3.3 (1H, dd,
J = 13.4, 5.3 Hz), 3.02 (6H, s), 2.93 (1H, m), 2.26 (1H, m), 2.16
(2H, m), 2.02 (1H, m), 1.64 (2H, m), 1.32 (3H, d, J = 4.9 Hz). 13C
NMR (150 Hz, CD3OD) δ (ppm) 175.96, 175.33, 172.46, 163.74,
151.14, 148.96, 147.43, 139.53, 131.90, 129.83, 126.42, 110.20,
109.18, 107.40, 101.91, 100.65, 99.63, 80.39, 74.55, 73.95, 71.55,
71.29, 68.43, 67.82, 66.46, 56.43, 55.35, 43.90, 43.15, 40.82, 38.0,
32.86, 32.59, 19.93, 19.39. HRMS (ESI, MicrOTOF) calcd for
C38H45NO17 787.2687, found 788.2432 (M þ 1). Etoposide 40-
dimethylglycine 300-hemiglutarate (8), 1H NMR (600 Hz,
CD3OD) δ (ppm) 7.0 (1H, s), 6.54 (1H, s), 6.38 (2H, s), 5.98
(2H, q, J = 4.0 Hz), 5.09 (1H, t, J = 9.2 Hz), 5.05 (1H, d, J = 3.2
Hz), 4.76 (1H-300, d, J = 7.6 Hz), 4.73 (1H, q, J = 5.1 Hz), 4.66
(1H, d, J = 5.6 Hz), 4.44 (1H, dd, J = 10.5, 8.8 Hz), 4.30 (1H, t.
J = 8.0 Hz), 4.18 (1H, dd, J = 10.3, 4.5 Hz), 3.67 (6H, s), 3.58
(2H, s), 3.57 (m, 1H), 3.48 (1H, dd, J = 14.0, 5.5 Hz), 3.44 (1H-
200, t, J = 10.2 Hz), 3.43 (1H, t, J = 9.4 Hz), 3.41 (1H, dd, J =
9.4, 4.1 Hz), 2.98 (1H, m), 2.47 (6H, s), 2.43 (2H, t, J = 7.0 Hz),
2.35 (2H, t, J = 7.4 Hz), 1.64 (2H, m), 1.32 (3H, d, J = 4.9 Hz).
13C NMR (150 Hz, CD3OD) δ (ppm) 176.27, 173.01, 167.79,
151.43, 148.94, 147.38, 139.21, 132.40, 129.21, 109.96, 109.78,
107.48, 102.28, 101.76, 99.53, 78.38, 73.75, 73.19, 72.64, 68.57,
67.87, 66.30, 58.81, 55.29, 44.06, 43.93, 40.98, 38.08, 33.16,
32.85, 20.49, 19.35. HRMS (ESI, MicrOTOF) calcd for
C38H45NO17 787.2687, found 788.2432 (M þ 1).
In Situ Mouse Brain Perfusion. The transport of [125I]-2, [125I]-
3, and [14C]-doxorubicin or [3H]-etoposide in mouse brain was
measured using the in situ brain perfusion method adapted in
our laboratory for the study of drug uptake in the mouse brain
(Dagenais et al.24). In situ brain perfusion of [14C]-inulin in the
presence of unlabeled 2 and 3 was also performed to verify the
physical integrity of the BBB. Briefly, the right common carotid
of mice anaesthetized with ketamine/xylazine (140/8 mg kg-1
,
ip) was exposed and ligated at the level of the bifurcation of the
common carotid, rostral to the occipital artery. The common
carotid artery was then catheterized rostrally with polyethylene
tubing filled with heparin (25 U/mL) and mounted on a
26-gauge needle. The syringe containing the perfusion fluid
(radiolabeled molecules at the appropriate concentrations in
Krebs/bicarbonate buffer (128 mM NaCl, 24 mM NaHCO3,
4.2 mM KCl, 2.4 mM NaH2PO4, 1.5 mM CaCl2, 0.9 mM MgCl2
and 9 mM D-glucose) gassed with 95% O2 and 5% CO2 to obtain
a pH of 7.4, and warmed to 37 °C in a water bath) was placed
in an infusion pump (Harvard pump PHD 2000; Harvard
Apparatus, Saint-Laurent, QC, Canada) and connected to
the catheter. Prior to the perfusion, the contralateral blood
flow contribution was eliminated by severing heart ven-
tricles. The brain was perfused for 5 min at a flow rate of
1.15 mL/min. After perfusion, the brain was further perfused
for 60 s with Krebs buffer to wash out the excess of radiolabeled
molecules. Mice were then decapitated to terminate perfusion
and the right hemisphere was quickly isolated on ice before
being subjected to capillary depletion. Briefly, for capillary
depletion, the mice brain was homogenized on ice in Ringer’s
HEPES buffer with 0.1% BSA in a glass homogenizer. Brain
homogenate was then mixed thoroughly with 35% Dextran 70
(50:50) and centrifuged at 5400g for 10 min at 4 °C. The
supernatant composed of brain parenchyma and the pellet
Synthesis of 3. DIEA (0.17 mL, 0.98 mmol) was added
dropwise to a mixture of 7 (330 mg, 0.42 mmol) and TBTU
(145 mg, 0.46 mmol) in DMF (24 mL). The mixture was stirred
at room temperature for 50 min, then a solution of Angpep-2
(422 mg, 0.14 mmol) in DMSO (1.5 mL) and DMF (9 mL) was
added, followed by DIEA (0.084 mL, 0.48 mmol). The mixture
was stirred at room temperature for 20 min. An aliquot (10 μL)
was taken for UPLC analysis, and it showed the reaction was
complete. After stirring for another 10 min, the reaction solution
was concentrated to 3 mL and purified using AKTA RPC
column (gradient 10- 25% MeCN in H2O with 0.05% FA).
After lyophilization, 3 (172 mg, 26%) was yielded as a colorless
powder. LC-HRMS (ESI, MicrOTOF), m/z calcd for C218H281
-
N32O79 4610.8955, found 2305.9327 (2þ), 1537.6443 (3þ),
1153.7463 (4þ), 922.7970 (5þ).
Iodination of 2 and 3 Derivatives. 2 and 3 were radiolabeled
with standard procedures using an Iodo-beads kit. A ratio of
two Iodo-beads per iodination was used for the labeling. Briefly,
beads were washed twice with 1 mL of PBS on a Whatman filter
and resuspended in 60 μL of PBS, pH 6.5. [125I]Na (1 mCi) was
added to the bead suspension for 5 min at room temperature.
Iodination of peptide derived anticancer drugs was initiated by
the addition of 250 μg of 2 or 3 (100-150 μL) diluted in 0.1 M

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2823
representing capillaries were then carefully separated. Aliquots
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125I]-3 samples were counted in a Wizard 1470 automatic γ
counter (Perkin-Elmer Inc., Woodbridge, ON). [3H]-Etoposide
and [14C]-doxorubicin samples were digested in 2 mL of Solva-
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[
125I]2 and [125I]3 tissue samples were counted in a Wizard 1470
automatic γ counter (Perkin-Elmer Inc., Woodbridge, ON).
[3H]-etoposide and [14C]-doxorubicin tissue samples were di-
gested in 2 mL of Solvable (Packard, Boston, MA) at 50 °C and
mixed with 9 mL of Ultima gold XR scintillation cocktail
(Packard, Boston, MA). Radioactivity was counted in a Pack-
ard Tri-Carb liquid scintillation counter model 1900 TR.
ꢀ
Acknowledgment. We thank Julie Poirier, Isabelle Lavallee,
and Tran Nguyen for their technical support. This work was
supported by research funding from the National Research
Council of Canada’s Industrial Research Assistance Program
(NRC-IRAP) to Angiochem, Inc. and a grant from the
Natural Sciences and Engineering Research Council of Canada
ꢀ
(NSERC) to R. Beliveau. The authors also thank David
Norris, PhD for editorial assistance provided during the
preparation of this manuscript. This assistance was paid for
through funding supplied by Angiochem, Inc. to Ecosse
Medical Communications, LLC (Princeton, NJ). Disclosure:
Angiochem, Inc., a biotechnology company located in
Montreal, Canada, developed the EPiC drug platform de-
scribed in this report. M.D. is an employee of Angiochem,
Inc., has shares and stock options in the company, and is listed
on company patent applications. C.C., G.Y., C.T., A.R., and
J.-P.C. are employees, have stock options in the company, and
are listed on company patent applications. M.-C.L. is also an
employee and has stock options in the company. R.B. has
received research funding and consulting fees from Angio-
chem, Inc., has shares in the company, and is listed on com-
panypatent applications. J.-C.C. reportsnofinancial conflicts
of interest pertaining to this work.
(22) ETOPOPHOS;Etoposide Phosphate Injection, Powder, Lyophi-
lized, for Solution [package insert]; Bristol-Myers Squibb Company:
Princeton, NJ, 2005.
(23) Doxorubicin Hydrochloride for Injection [package insert]; Pfizer Inc.:
New York, 2006.
(24) Dagenais, C.; Rousselle, C.; Pollack, G. M.; Scherrmann, J. M.
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