Improved therapeutic efficacy of doxorubicin through conjugation with a novel peptide drug delivery technology (Vectocell)

Article abstract of DOI:10.1021/jm0606591

Improvement in the therapeutic index of doxorubicin, a cytotoxic molecule, has been sought through its chemical conjugation to short (15-23 amino acid) peptide sequences called Vectocell peptides. Vectocell peptides are highly charged drug delivery peptides and display a number of characteristics that make them attractive candidates to minimize many of the limitations observed for a broad range of cytotoxic molecules. The studies reported here characterized the in vitro and in vivo efficacy of a range of Vectocell peptides conjugated to doxorubicin through different linkers. These studies show that the in vivo therapeutic index of doxorubicin can be improved by conjugation with a specific Vectocell peptide (DPV 1047) through an ester linker to C14 of doxorubicin, in both colon and breast tumor models. This conjugate was also shown to have significant in vivo antitumoral activity in a model resistant to doxorubicin, suggesting that this conjugate is able to circumvent the multidrug resistance (MDR) phenotype. These experiments therefore provide support for the use of the Vectocell technology with other cytotoxic agents.

Full text of DOI:10.1021/jm0606591

6908
J. Med. Chem. 2006, 49, 6908-6916
Improved Therapeutic Efficacy of Doxorubicin through Conjugation with a Novel Peptide Drug
Delivery Technology (Vectocell)
Florence Meyer-Losic,* Je´roˆme Quinonero, Vincent Dubois, Bertrand Alluis, Mireille Dechambre, Matthieu Michel,
Franc¸oise Cailler, Anne-Marie Fernandez, Andre´ Trouet, and Jonathan Kearsey
Diatos S.A., 166 BouleVard du Montparnasse, 75014 Paris, France
ReceiVed June 2, 2006
Improvement in the therapeutic index of doxorubicin, a cytotoxic molecule, has been sought through its
chemical conjugation to short (15-23 amino acid) peptide sequences called Vectocell peptides. Vectocell
peptides are highly charged drug delivery peptides and display a number of characteristics that make them
attractive candidates to minimize many of the limitations observed for a broad range of cytotoxic molecules.
The studies reported here characterized the in vitro and in vivo efficacy of a range of Vectocell peptides
conjugated to doxorubicin through different linkers. These studies show that the in vivo therapeutic index
of doxorubicin can be improved by conjugation with a specific Vectocell peptide (DPV1047) through an
ester linker to C14 of doxorubicin, in both colon and breast tumor models. This conjugate was also shown
to have significant in vivo antitumoral activity in a model resistant to doxorubicin, suggesting that this
conjugate is able to circumvent the multidrug resistance (MDR) phenotype. These experiments therefore
provide support for the use of the Vectocell technology with other cytotoxic agents.
Introduction
rather than to directly inhibit MDR mechanisms, by developing
anticancer therapies that are not substrates for extracellular
export.
The objective of this research was to assess the in vivo
therapeutic benefit of novel peptidic-doxorubicin conjugates
in both drug-sensitive and drug-resistant cancer models. Vec-
tocell peptides (or DPV for Diatos Peptide Vectors) are short
(15-23 amino acid) peptide sequences that are able to transport
molecules across the cell membrane via endocytosis and have
been developed as tools for intracellular delivery of a broad
range of therapeutic molecules.1-3 These peptides originate from
human heparin binding proteins and/or anti-DNA antibodies and,
once conjugated to a therapeutic molecule, can selectively
deliver the molecule to either the cytoplasm or the nuclei of
mammalian cells.4,5
The conjugation of Vectocell peptides to cytotoxic molecules
can modify the in vivo distribution of the therapeutic molecules,
improving pharmacokinetic properties and/or reducing systemic
toxicity by driving tissue and intracellular delivery.2,5 In addition,
conjugation of Vectocell peptides to small molecules/drugs may
also provide a means of inhibiting the extracellular export of
the therapeutic agents by proteins involved in multidrug
resistance (MDR).6
Multidrug resistance can seriously limit cancer chemotherapy
treatment, for example, through the overexpression of membrane
transporters that mediate unidirectional energy-dependent drug
efflux, thus reducing intracellular drug levels. These membrane
transporters are normally expressed in high levels within cells
involved in detoxification, such as the liver, kidney, and colon.7,8
Tumors arising from these cells are often resistant to chemo-
therapy treatment from their onset, while other tumors can
acquire resistance through the induction of MDR transport
proteins during treatment.8-10 Many inhibitors of MDR trans-
porters have been identified. However, these inhibitors also
interfere with the clearance of the cytotoxic drug, resulting in
elevated plasma concentrations of the cytotoxic agent and
associated toxicity.11 An alternative approach is to circumvent
Doxorubicin, an anthracycline antibiotic, remains among the
most widely used cytotoxic agents for the treatment of a broad
spectrum of cancers, including breast, stomach, non-Hodgkin’s
lymphoma, and bladder cancer.12,13 As with many cytotoxic
drugs, doxorubicin has severe short- and long-term side effects,
in this case mostly associated with bone marrow and myocardial
cell toxicity.12,14 Cardiotoxicity limits the cumulative dosage
of doxorubicin to 500-600 mg/m2, which may be a dose at
which tumor is still responding to treatment but for which no
further doxorubicin treatment can be given.15,16 Another draw-
back of doxorubicin is the emergence of drug resistance that
results in the reduction of the intracellular concentration of
doxorubicin.17
The present study aimed to generate novel peptidic-doxo-
rubicin conjugates by use of three Vectocell peptides that differ
in terms of their charge, size, and intracellular delivery
characteristics and to assess their ability to enhance the
therapeutic potential of doxorubicin and to prevent the appear-
ance of MDR. Different conjugation sites and linkers of different
stabilities were used to generate Vectocell-doxorubicin con-
jugates in order to evaluate their effect on efficacy. Chemical
routes were developed to allow the conjugation of doxorubicin
to Vectocell peptides through ester, thioether, and amide
chemical linkers. The ester and thioether involved carbon 14
of doxorubicin, and the amide carbon 3′. In vitro and in vivo
characterization has defined the optimal conjugate-linker
combination that significantly increases efficacy above uncon-
jugated doxorubicin in both doxorubicin-sensitive and -resistant
models. The data presented therefore provide in vivo proof of
concept for the use of Vectocell peptides to improve the
therapeutic index of doxorubicin, and potentially many other
cytotoxic or small molecule anticancer drugs.
Results
* Corresponding author: Research Division, Diatos S. A. Laboratories,
11-13 rue Watt, 75013 Paris, France; tel +33 (0)1 53 80 93 55; fax +33
(0)1 53 80 93 89; e-mail fmeyerlosic@diatos.com.
Chemistry. The synthesis of three different types of doxo-
rubicin conjugates covalently attached to Vectocell peptides has
10.1021/jm0606591 CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/12/2006

Vectocell-Doxorubicin Therapeutic EValuation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6909
etry, as an acetone adduct of 14-doxorubicinyl maleimidobu-
tyrate. The byproduct obtained is not reactive in the following
step and can be removed by extraction.
The next step consists of the coupling of 14-doxorubicinyl
maleimidobutyrate (6) via its maleimido moiety with the thiol
group of the peptide (7a-c) (see Experimental Section and
Scheme 2), resulting in the desired peptide-doxorubicin ester
conjugate (8a-c). A second route was investigated that consists
of reacting halogenated daunorubicin (3) directly with the thiol
moiety of the peptide (7a-c) as described in Scheme 2, thereby
obtaining a more stable thioether conjugate (9b-c).
Doxorubicin peptide conjugates were also prepared from
doxorubicin, in which the peptide is coupled on the 3′ amino
group of the daunosamine sugar of the anthracycline (10)
(Scheme 3). After conjugation of maleimidobutyric acid (4) to
the amino group of doxorubicin (10), the activated anthacycline
(11) was conjugated with the peptide (7a,b) through the thiol
group as described above. Thus, peptide-doxorubicin amide
conjugates (12a,b) were obtained.
Figure 1. Acetone adduct byproduct of the 14-halodaunorubicin
esterification.
been undertaken. Ester or thioether bonds have been used to
attach the Vectocell peptide at position 14 (C14, Figure 1), which
should not inhibit the interaction of doxorubicin with DNA. The
3′ amino group (Figure 1) was used to conjugate the Vectocell
peptide via an amide bond, which has the potential of inhibiting
interaction of the amine group with DNA.12,13
One Vectocell-doxorubicin conjugation route has been
suggested by the work of Horton et al.,18 who described the
synthesis of doxorubicin and 14-O-acetyldoxorubicin from
daunorubicin via 14-bromodaunorubicin. This chemical route
(Scheme 1) was shown to allow the generation of the desired
doxorubicin-peptide conjugate, through either an ester bond
(cleavable) or a thioether bond (noncleavable).
The first step of this synthesis (Scheme 1) was the halogena-
tion of daunorubicin hydrochloride (1) on its 14 position19 via
the 14-bromo-13-dimethylketal-daunorubicin (2) intermediate,
which was directly hydrolyzed (without purification) to 14-
halodaunorubicin (3) (mixture of 14-bromo- and 14-chlo-
rodaunorubicin) rather than the expected 14-bromodaunorubicin
(Scheme 2) due to the use of sodium chloride during the
extraction step (see Experimental Section). Halogen exchange
can be prevented by using saturated sodium bromide instead of
sodium chloride during this step. Analysis of the acetal
intermediate showed the presence of 14-chlorodaunorubicin (9%
of peak area), presumably arising because the daunorubicin
starting material was a hydrochloride salt. It was possible to
form exclusively 14-bromodaunorubicin by avoiding the pres-
ence of chloride ions.
Selection of the Optimal Conjugate. The Vectocell peptides
(7a, 7b, and 7c; DPV1047, DPV10, and DPV7b, respectively)
were conjugated to doxorubicin (10) via the three types of
chemical bond described above, and the in vitro cytotoxicity
of the synthesized compounds was evaluated in two doxorubi-
cin-sensitive (HCT116, MCF7/6) tumor cell lines (Table 1). The
Vectocell peptides alone showed no cytotoxic activity (data not
shown2). The Vectocell-doxorubicin conjugates 8a-c showed
no loss of cytotoxicity compared to free doxorubicin. Com-
pounds 9b and 9c showed a loss of activity in the HCT116
model but not in the MCF-7/6 cell model, compared to free
doxorubicin (10). In contrast, the compounds 12a and 12b
showed a significant loss of cytotoxicity in both tumor cell lines
(loss of 1-2 logs in IC50). The in vitro data, therefore, suggest
that the ester (8a-c) and thioether (9b and 9c) bonds are optimal
for Vectocell-doxorubicin conjugate activity and that conjuga-
tion to the 3′ position via an amide bond (12a and 12b) partially
inactivates doxorubicin.
The in vitro cytotoxicity of the conjugates was also tested in
a doxorubicin-resistant cell model (MCF7-Adr, Table 1). These
experiments showed that it is possible to overcome the doxo-
rubicin-resistant phenotype by conjugation of doxorubicin to
Vectocell peptides. Vectocell peptides DPV1047 (8a and 12a)
and DPV10 (8b and 9b) are able to inhibit the doxorubicin-
14-Halodaunorubicin (3) (Scheme 2) was then acylated by
use of sodium maleimidobutyrate (5) [previously obtained by
treatment with sodium hydrogen carbonate of the parent acid
(4)] to generate 14-doxorubicinyl maleimidobutyrate (6). One
major impurity was formed and identified, by mass spectrom-
Scheme 1. Synthesis of Doxorubicin-Peptide Conjugates Starting from Daunorubicina
a Reagents and conditions: (i) Trimethyl orthoformate, methanol, dioxane, Br2, and then propylene oxide and diisopropyl ether; (ii) 48% HBr, acetone,
and then NaCl.

6910 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Meyer-Losic et al.
Scheme 2. Synthesis of Doxorubicin-Peptide Conjugates in C14 Positiona
a Reagents and conditions: (i) Trimethyl orthoformate, methanol, dioxane, Br2, and then propylene oxide and diisopropyl ether; (ii) 48% HBr, acetone,
and then NaCl; (iii) NaHCO3, H2O; (iv) acetone, refluxed; (v) H2O and then CH2Cl2, DMF; (vi) methanol, K2CO3, and then Tris-HCl buffer pH 9, CHCl3,
wherein R1 ) -OH or -NH-peptide; R2 ) -H or -CO-peptide.
resistant phenotype of MCF7-Adr cells. However, the com-
pounds 8a and 9b exhibited the greatest cytotoxic activity in
the doxorubicin-resistant cell model, although 9b showed a
loss of cytotoxic activity in the HCT116 in vitro model (Table
1). The in vitro data therefore suggested that the optimal
conjugate for both doxorubicin-sensitive and -resistant models
was 8a.
The in vivo antitumoral efficacy of the different conjugates
was compared in the human HCT116 colon carcinoma model
(doxorubicin-sensitive) administered by intravenous injection
(iv) (Table 1). The conjugates 8a-c all showed activity; the
most active was 8a. Compound 12a showed no activity and
confirmed the in vitro data. In a second experiment, conjugates
9c, 9b, and 8a were compared. Compound 9c was completely
inactive and 9b was only partially active, which correlated with
the reduction of in vitro cytotoxicity of both 9c and 9b compared
to 8a observed in the HCT116 model. The in vivo evaluation
of the Vectocell-doxorubicin conjugates confirmed that 8a (15
µmol/kg) is the optimal conjugate with a T/C value of 29.5%
(Table 1), whereas T/C obtained with the other conjugates
ranged from 34.1% (8c) to 86.2% (9c). Moreover, in this
experiment, the conjugate 8a (15 µmol/kg) showed better
efficacy than doxorubicin (10, 6.5 µmol/kg: doxorubucin’s
maximal tolerated dose, MTD), with a T/C of 49.6%. It should
be noted that it is possible to administer 8a at twice the MTD
of doxorubicin (10), demonstrating that 8a is less toxic and more
active than 10. For this reason 8a was therefore selected for
further preclinical evaluation in both doxorubicin-sensitive and
-resistant models.
Extensive Evaluation of 8a. Experiments were undertaken
to evaluate whether conjugation of the Vectocell peptide to
doxorubicin was required to enhance activity. Compounds 10

Vectocell-Doxorubicin Therapeutic EValuation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6911
Scheme 3. Synthesis of Doxorubicin-Peptide Conjugates with an Amide Linka
a Reagents and conditions: (i) DIPEA, HATU, DMF; (ii) H2O and then CH2Cl2, DMF; wherein R1 ) -OH or -NH-peptide; R2 ) -H or -CO-peptide.
Table 1. In Vitro and in Vivo Selection of Vectocell-Doxorubicin Conjugatesa
in vivo efficacy (HCT116)
compd
composition
doxorubicin
HCT116 [S] (µM)
MCF-7/6 [S] (µM)
MCF-7/Adr [R] (µM)
T/C (%)/dose (µmol/kg)
10
8a
8b
8c
9b
9c
12a
12b
3
6
2
18
29
9
10
37
17
108
114
> 1000
133
532
> 1000
49.6/6.5
29.5/15
39/15
34.1/15
55.8/10
86.2/10
79.7/15
nd
DPV1047-E-Dox
DPV10-E-Dox
DPV7b-E-Dox
DPV10-TE-Dox
DPV7b-TE-Dox
DPV1047-A-Dox
DPV10-A-Dox
5
153
30
nd
200
121
862
500
625
a In vitro data: Mean IC50 values (micromolar) from three or more independent experiments are given. [S] Doxorubicin-sensitive cell line; [R] doxorubicin-
resistant cell line. In vivo HCT116 experiment: NMRi nude mice were intradermally grafted with HCT116 tumor cells. Mice were treated when tumors
were established by iv injections (three injections per week for 3 weeks) with the different compounds. Conjugates were administered at their maximal
administrable dose (MAD); doxorubicin was administered at its maximal tolerate dose (MTD). Tumor volume was evaluated and T/C was calculated.
Table 2. Confirmation That Chemical Conjugation of DPV1047 to
The in vivo antitumor activity of 8a was then evaluated in
Doxorubicin is Required to Increase the in Vitro Cytotoxicity of
the MDA-MB-231 human breast adenocarcinoma model (Figure
2A). In this study, DPV1047-E-doxorubicin conjugate (8a) was
Doxorubicina
HCT116 [S]
MCF-7/Adr [R]
shown to have a significant dose-dependent efficacy, with a
optimal T/C of 27.6% at 8 µmol/kg (p < 0.001) and a optimal
T/C of 16.4% at 10 µmol/kg. Moreover, at the higher dose 8a
treatment induced three transient and one complete regression.
At an equitoxic dose [as shown by the same maximum body
weight loss (Figure 2B)], 8a was also found to be more active
than doxorubicin (10), with optimal T/C values of 43.4% for
doxorubicin (10) at 6 µmol/kg and 16.4% for 8a at 10 µmol/kg
(p < 0.001, Figure 2A). The improved efficacy was still
significant at 8 µmol/kg 8a, (T/C ) 27.6%, p < 0.001)
compared to doxorubicin (10) at 6 µmol/kg.
The DPV1047-E-doxorubicin conjugate (8a) was then
compared to doxorubicin (10) in the HCT15 human colorectal
adenocarcinoma model (Figure 3A). This cell line has been
shown to constitutively express P-glycoproteins (Pgps) and was
thus chosen for its partial resistance to doxorubicin.20 The in
vivo expression of Pgp in this tumor model was confirmed by
immunohistochemistry and showed a high level of immunore-
activity in the membrane of the tumor cells (not shown). In
compd
doxorubicin (10)
DPV1047 (7a)
doxorubicin (10) + DPV1047 (7a)
(nonconjugated)
(µM)
(µM)
3
>1000
4
>1000
>1000
>1000
DPV1047-E-Dox (8a, conjugated)
7
260
a Mean IC50 values (micromolar) from three or more independent
experiments are given. [S] Doxorubicin-sensitive cell line; [R] doxorubicin-
resistant cell line.
(doxorubicin) and 7a (DPV1047) were coincubated (without
chemical conjugation) with both doxorubicin-sensitive and
-resistant cell lines (Table 2). Administration of the Vectocell
peptide (7a) alone has no antitumoral activity in vitro. In the
doxorubicin-resistant cell line, coadministration of 10 and 7a
showed no antitumoral activity, confirming that conjugation of
the Vectocell peptide to doxorubicin is required to elicit the
cytotoxic effect in this doxorubicin-resistant cell line [as
demonstrated in Table 2 by the activity of 8a (DPV1047-E-
Dox) on MCF7-Adr cells].

6912 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Meyer-Losic et al.
Figure 3. Antitumoral efficacy (A) and toxicity (B) of DPV1047-
E-Dox (8a) and doxorubicin (10) in the doxorubicin-resistant HCT15
human colon cancer model. Compounds were administered three times
a week for 3 weeks (see arrowheads, 1). (s) Vehicle-treated animals;
(--9--) 10 (doxorubicin), 4.8 µmol/kg; (-9-) 10 (doxorubicin), 6
µmol/kg; (--2--) 8a (DPV1047-E-Dox), 8 µmol/kg; (-2-) 8a
(DPV1047-E-Dox), 10 µmol/kg. *p < 0.05 (in comparison with
doxorubicin).
Figure 2. Antitumoral efficacy (A) and toxicity (B) of DPV1047-
E-Dox (8a) and doxorubicin (10) in MDA-MB-231 human breast
cancer model. Compounds were administered three times a week for
3 weeks (see arrowheads, 1). (s) Vehicle-treated animals; (-9-)
10 (doxorubicin), 6 µmol/kg; (--2--) 8a (DPV1047-E-Dox), 8
µmol/kg; (-2-) 8a (DPV1047-E-Dox), 10 µmol/kg. *p < 0.05,
**p < 0.01 (in comparison with doxorubicin).
Table 3. DPV1047-E-Dox (8a) and Doxorubicin (10) Cytotoxicity in
Three Doxorubicin-Resistant Human Cell Linesa
this partially resistant, Pgp-expressing, tumor model (Figure 3)
the DPV1047-E-doxorubicin conjugate (8a) displayed a sig-
nificantly (p < 0.05) better antitumoral efficacy than free
doxorubicin (10). The body weight losses induced by both drugs
at these doses were similar, indicating equitoxicity (Figure 3B).
In this experiment, the dose-related efficacy was evaluated by
administering a dose 20% inferior to the MTD of both drugs.
Reducing the optimal dose of doxorubicin (10) or 8a by 20%
significantly reduced the efficacy of doxorubicin (10, optimal
T/C of 50% vs 38.6%), whereas the antitumoral activity of the
DPV1047-E-doxorubicin conjugate (8a) was maintained (op-
timal T/C of 26.6% vs 24.7%, Figure 3A).
In Vitro Inhibition of Multidrug Resistance. To further
explore the ability of the DPV1047-E-doxorubicin conjugate
(8a) to circumvent MDR, its cytotoxicity was correlated with
the expression of the MDR protein Pgp (P-glycoprotein) in three
doxorubicin-resistant cell lines (Table 3). In these resistant cell
lines, DPV1047-E-Dox (8a) always showed better antiprolif-
erative activity than doxorubicin. In MCF7-Adr and MES-SA-
dx5 cells, which express high levels of Pgp, the enhanced
efficacy of DPV1047-E-Dox (8a) was highly significant
compared to that of doxorubicin alone (p < 0.001).
HCT15 MES-SA/dx5 MCF-7/Adr
Pgp expressionb
doxorubicin (µM)
DPV1047-E-Dox (8a) (µM)
5
283
50
22
600
81***
28
>1000
260***
a Mean IC50 values (micromolar) from three or more independent
experiments are given. ***p < 0.001 in comparison with doxorubicin.
b Mean fluorescence intensity by flow cytometry.
increase in sensitivity was observed, with only a 3.6-5-fold
increase in activity. The difference between doxorubicin (10)
and DPV1047-E-Dox (8a) cytotoxicity following Vp treatment
suggests that DPV1047-E-Dox (8a) is a poor substrate for Pgp-
mediated cell extrusion, which could result in an increase in
the intracellular concentration of the therapeutic molecule.
Discussion
The search for better cancer therapies relies on either
identifying new therapeutic molecules with new mechanisms
of action or improving the therapeutic index of chemotherapeutic
agents that are already available. The goal of this study was to
evaluate the capacity of Vectocell peptides to improve the
therapeutic index of doxorubicin (10), a drug that still remains
one of the most prescribed drugs for cancer treatment.12
Vectocell peptides are polycationic cell-penetrating peptides
(15-23 amino acid residues) originating from human proteins.
Vectocell peptides have been extensively studied by de Coupade
et al.2 and were shown to be internalized by different mecha-
nisms, delivered to different cellular compartments, and have
To verify that DPV1047-E-Dox is able to circumvent the
action of Pgp drug pumps, experiments were performed with
an inhibitor of Pgp function, verapamil (Vp).8,21 Vp was added
to cultured cells prior to the cytotoxic compound and was able
to increase the sensitivity of the resistant cells to doxorubicin
(10) between 11- and 33-fold (Table 4). In the case of Vp
followed by DPV1047-E-Dox (8a) treatment, only a moderate

Vectocell-Doxorubicin Therapeutic EValuation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6913
Table 4. Effect of Pgp Expression on Cytotoxic Activity of DPV1047-E-Dox (8a) and Doxorubicin (10)a
HCT116 [S] (µM)
HCT15 [R] (µM)
MCF-7/Adr [R] (µM)
MES-SA/dx5 [R] (µM)
doxorubicin (10)
alone
+ 20 µM Vp
alone
4
2
5
4
228
20
50
> 1000
30
600
21
79
DPV1047-E-Dox (8a)
260
52
+ 20 µM Vp
11
22
a Mean IC50 values (micromolar) from three or more independent experiments are given. [S] Doxorubicin-sensitive cell line; [R] doxorubicin-resistant
cell line. Vp, verapamil.
different intracellular delivery levels. The internalization of
Vectocell peptides occurs following their interaction with cell
surface glycosaminoglycans (GAGs, polyanionic molecules
present on the surfaces of all adherent animal cells that interact
with many families of growth factors and growth factor receptors
and influence cell growth and differentiation) via energy-
dependent caveolar endocytosis, as has been described for other
cell-penetrating peptides.2 One particular peptide, DPV1047
(7a), that partially originates from an anti-DNA antibody is
internalized in a caveolar-independent manner. In the present
study, Vectocell peptides that show different internalization
characteristics have been linked to doxorubicin to assess in vitro
and in vivo efficacy.
A number of doxorubicin-peptidic conjugates have been
reported in the literature,22-24 and all have been shown to require
peptide cleavage in order to reactivate doxorubicin. It was,
however, hypothesized that the use of a positively charged
Vectocell peptide could stabilize the doxorubicin interaction with
negatively charged DNA, allowing improved activity, suggesting
that a stable chemical linker may enhance activity. This
hypothesis was evaluated by the conjugation of Vectocell
peptides to different positions on doxorubicin by labile or
nonlabile chemical bonds.
The selection of an optimal chemical bond and conjugation
position was found to be important for increased activity. In
contrast with the initial expectation, the use of chemically stable
bonds for conjugation of the peptide at position 14 (thioether)
or at position 3′ (amide) of doxorubicin (10) resulted in
significant or complete loss of activity in vivo, presumably
because the Vectocell peptides inhibit the interaction of doxo-
rubicin (10) with DNA. A relatively labile chemical bond,
allowing doxorubicin release, is therefore required, and for this,
the C14 position and an ester bond are ideal. In fact, the ester
bond was shown to be partially labile in plasma in vivo, with
both the ester conjugate [DPV1047-E-Dox (8a)] and the
metabolite doxorubicin being present in the blood 30 min after
DPV1047-E-Dox (8a) injection (the ratio of 8a/doxorubicin
at 30 min post-8a injection is approximately 0.1, data not
shown). This confirmed that the ester link is labile but that it is
sufficiently stable to allow DPV-driven intracellular delivery
and improve in vivo efficacy compared to free doxorubicin.
As expected, the choice of Vectocell peptide also affected
the in vitro and in vivo efficacy of the conjugate. DPV1047
and DPV10 ester conjugates (8a and 8b, respectively) had an
improved cytotoxic activity on resistant cell lines compared to
free doxorubicin, which was not the case for the DPV7b ester
conjugate (8c). In the in vivo models that are sensitive to
doxorubicin (10), the use of 8a (DPV1047-E-Dox), 8b, and
9c shows a clear benefit over the use of doxorubicin (10) alone.
This benefit was not, however, observed in the in vitro
cytotoxicity data (Table 1), which suggests that 8a (DPV1047-
E-Dox), 8b, and 9c alter the in vivo biodistribution and/or
pharmacokinetic properties of doxorubicin (10), thereby reduc-
ing the exposure of normal tissues to doxorubicin and increasing
its concentration in the tumor (10). This appears to be the case
as DPV1047-E-Dox (8a) reduced the systemic toxicity of
doxorubicin (10) and at equitoxic doses it exhibits significantly
improved antitumoral efficacy compared to doxorubicin (10)
alone in three different in vivo tumor models.
The present study suggests that conjugation of doxorubicin
to a Vectocell peptide allows circumvention of the MDR
phenotype. The mechanism of Vectocell-doxorubicin internal-
ization may deliver doxorubicin to intracellular compartments
that are less accessible to Pgps. During the preparation of this
paper, Liang and Yang25 have confirmed this observation using
a TAT (CGGGYGYGRKKRRQRRR)-doxorubicin conjugate.
The TAT-doxorubicin conjugate published by Liang and Yang
corresponds most closely to compounds 12a,b, conjugates that
were active but not the optimal molecule selected in our studies.
The ability of DPV1047-E-Dox (8a) to increase the efficacy
and reduce the systemic toxicity of doxorubicin (10) suggests
not only that DPV1047-E-Dox (8a) will be useful in indications
for which doxorubicin (10) is currently used, but also that
DPV1047-E-Dox (8a) may be applied to indications that are
currently refractory to doxorubicin treatment. The mechanism
of DPV1047-E-Dox (8a) intracellular delivery, although not
fully characterized, appears to be different from that of other
Vectocell or cell-penetrating peptides such as TAT.2 This may
be, in part, due to the lower charge-to-mass ratio observed for
DPV1047 compared to other cell-penetrating peptides, which
in turn results in an improved therapeutic index.
In conclusion, our results demonstrate that this novel doxo-
rubicin conjugate, DPV1047-E-Dox (8a, subsequently to be
named DTS-101), shows increased antitumoral efficacy against
doxorubicin-sensitive and -resistant tumor models. In vitro and
in vivo data indicate that doxorubicin (10) delivered in the form
of DPV1047-E-Dox (8a) has a significantly higher activity than
doxorubicin (10) and suggests that DPV1047-E-Dox delivers
doxorubicin to an intracellular compartment that is less acces-
sible to the action of membrane Pgps. Our results also suggest
that the Vectocell technology could be used to increase the
therapeutic index of other cytotoxic and small molecule
anticancer drugs.
Experimental Section
Chemistry Reagents and Conditions. Daunorubicin hydro-
chloride and doxorubicin hydrochloride were obtained from Meiji
Seika Kaisha Ltd., Japan (these compounds and their derivatives
should be handled carefully due to their carcinogenic properties).
The peptide DPV1047 (7a, CVKRGLKLRHVRPRVTRMDV) was
produced by Neosystem, France, and the peptides DPV7b (7c,
GKRKKKGKLGKKRPRSRC) and DPV10 (7b, SRRARRSPR-
HLGSGC) were produced by Bachem, Switzerland.
Maleimidobutyric acid, n-butanol, absolute dioxane, and sodium
bicarbonate were obtained from Fluka, Switzerland. Acetone,
bromine, hydrobromic acid 48% in water, diisopropyl ether,
methanol, potassium carbonate, trifluoroacetic acid (TFA), trimethyl
orthoformate, diisopropylethylamine (DIPEA), and O-(7-azaben-
zotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HATU) were obtained from Aldrich, France. Acetic acid and tris-
(hydroxymethyl)aminomethane (Tris) were from Acros, Belgium.
Acetonitrile and dichloromethane were from SDS, France. Chlo-
roform and n-hexane were from Labscan, Ireland. Dimethylforma-

6914 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Meyer-Losic et al.
1
mide (DMF) was from Merck, United States. Propylene oxide was
from Janssen, United States, and sodium chloride was from Vel,
Belgium. HPLC analyses of all synthesized compounds were
performed at ambient temperature on a Agilent 1100 series system
equipped with a diode array and fluorescence detectors and using
Luna C18(2), 3 µm, 4.6 × 100 mm (Phenomenex) column with
0.1% TFA in water as solvent A, 0.1% TFA in acetonitrile as
solvent B, and a flow of 1.2 mL/min. Gradient elution conditions
were 5-60% B in 10 min, 60-90% B in 1 min, and 90% B for 3
min. The purity of the generated compounds was assessed as the
(5) was obtained as an off-white solid (9.06 g; 100% yield). H
NMR (250 MHz, D2O) δ 1.73 (q, 2H, H3′′, J ) 7.0 Hz); 2.10 (t,
2H, H2′′, J ) 7.4 Hz); 3.43 (t, 2H, H4′′, J ) 6.8 Hz); 6.73 (s, 2H,
H6′′). m/z (ESI): 182 [M - H]-.
14-Doxorubicinyl Maleimidobutyrate Ester (6). 14-Bro-
modaunorubicin/14-chlorodaunorubicin HBr salt, approximately 1/1
(3) (8.4 g; 12.6 mmol), and sodium 4-maleimidobutyrate (5) (13.5
g; 65.8 mmol) in acetone (1.2 L) were stirred and heated under
reflux and under argon, for 3 h. When cool, the mixture was filtered
through Whatman no. 40 filter paper, the solid was well washed
with acetone (total ca. 200 mL), and the filtrate was evaporated at
reduced pressure (water bath at 30 °C). The red residue (10.3 g)
was dissolved in water containing 0.1% TFA (340 mL) and
chromatographed on reverse-phase silica (YMC ODS-A, 500 g).
The column was eluted with a stepwise gradient of acetonitrile
(10%, 20%, 25%, 30%, and 50%) in water containing 0.1% TFA
and the fractions were checked by HPLC. The product fractions
were combined and the solvent was removed, initially in a rotary
evaporator (bath at 30-32 °C), and then on a freeze-drying unit.
The product (6) was obtained as a red solid (7.2 g; 69% yield)
containing one major impurity, which was isolated. This byproduct
was formed consecutively to the esterification reaction and identified
by mass spectrometry as an acetone adduct of doxorubicin
14-maleimidobutyrate. Compound 6: 1H NMR (250 MHz, CD3-
OD) 1.31 (d, 3H, H6′, J ) 6.4 Hz); 1.93 (m, 4H, H2′ and H3′′);
2.12 (br d, 1H, H8); 2.47 (m, 3H, H8 and H2′′); 2.98 (d, 1H, H10,
Jgem ) 18.5 Hz); 3.14 (d, 1H, H10, Jgem ) 18.5 Hz); 3.61 (m, 4H,
H3′, H4′, and H4′′); 4.05 (s, 3H, H15); 4.33 (q, 1H, H5′, J ) 6.4
Hz); 5.07 (m, 1H, H14, Jgem ) 17.7 Hz); 5.10 (s, 1H, H7); 5.31 (d,
1H, H14, Jgem ) 17.7 Hz); 5.46 (br s, 1H, H1′); 6.81 (s, 2H, H6′′);
7.60 (d, 1H, H3, J ) 8.2 Hz); 7.87 (t, 1H, H2, J ) 8.2 Hz); 8.01
(d, 1H, H1, J ) 7.5 Hz). m/z (ESI): 709 [M + H]+. Acetone
adduct: 1H NMR (250 MHz, D2O) 1.25 (d, 3H, H6′, J ) 5.2 Hz);
1.94 (m, 4H, H2′ and H3′′); 2.17 (s, 3H, H11′′); 2.56 (m, 4H, H8
and H2′′); 2.87 (m, 3H, H10 and H9′′); 3.12 (m, 4H, H10, H6′′,
and H7′′); 3.61 (m, 4H, H3′, H4′, and H4′′); 3.82 (s, 3H, H15);
4.24 (br s, 1H, H5′); 5.24 (m, 3H, H14 and H7); 5.43 (br s, 1H,
H1′); 7.30 (m, 2H, H3 and H2); 7.57 (m, 1H, H2). m/z (ESI): 767
[M + H]+.
Peptide-Doxorubicin Ester Conjugate (8a-c). General pro-
cedure: A solution of doxorubicin-14-maleimidobutyrate (6) (4.59
g; 3.91 mmol) in oxygen-free water (160 mL) was added at room
temperature under argon, in a glovebag, to a solution of peptide
(7a) (11.878 g, gross; 66% peptide content, 7.92 g, net; 3.27 mmol)
in oxygen-free water (160 mL). An additional 80 mL of water was
used to rinse the flask that contained 14-doxorubicinyl maleimi-
dobutyrate (6). The resulting solution was stirred at room temper-
ature under argon for 48 h, and then extracted 30 times with 10%
DMF in dichloromethane (100 mL), and three times with dichlo-
romethane alone (100 mL). The aqueous layer was lyophilized to
give 12.5 g of crude peptide conjugate as a red solid.
The solid was purified by means of preparative HPLC separation
with a Micromass ZMD instrument and a Luna C18(2), 10 µm,
250 × 21.2 mm semipreparative column (Phenomenex ref 00G-
4253-P0) with 0.1% TFA in water as solvent A and 0.1% TFA in
acetonitrile as solvent B (gradient, 5-30% B in 10 min, 3 min at
30% B, 30-90% B in 1 min, 6 min at 90% B; flow, 20 mL/min;
loading, 200 mg/run in 1 mL). For each injection, the desired
product was detected and collected by means of both the UV
response and the Fraction-Lynx mass detector system, set at m/z
) 783 for the [M + 4H]4+ ion of the desired conjugate. The relevant
fractions for each injection were combined and the resulting solution
was lyophilized to give doxorubicin-peptide conjugate (8a) TFA
salt as a red solid. A total of 5.35 g of material (52% yield) was
obtained from the 12.5 g of crude material. LC-MS for conjugate:
m/z 783 (M + 4H)4+, 1044 (M + 3H)3+, 96% purity (surface area,
HPLC). TFA analysis ) 26.9% (theory ) 26.7% for 10 molar equiv
CF3COOH). Compound 8b: 86% yield; m/z (ESI) 1202 (M +
2H)2+, 2403 (M + H)+; 97.6% purity (surface area, HPLC).
Compound 8c: 84% yield; m/z (ESI) 705 (M + 4H)4+, 940 (M +
3H)3+, 1410 (M + 2H)2+; 99.2% purity (surface area, HPLC).
percentage surface area of the peaks at 214 nm. 1H NMR and 13
C
NMR spectra were recorded on a Bruker FRX 250 and 400 MHz
spectrometer with tetramethylsilane as an internal standard. Chemi-
cal shifts (δ) are reported in parts per million (ppm) and J values
are quoted in hertz (Hz). 1H assignments were supported by (1H-
1H) COSY spectra. The splitting systems are as follows: s ) singlet,
d ) doublet, t ) triplet, m ) multiplet, br ) broad, br s ) broad
singlet, br d ) broad doublet, br t ) broad triplet, dd ) doublet
doublet, ddd ) doublet doublet doublet.
LC-MS analyses were performed with electrospray ionization.
Electrospray mass spectra (direct injections) were performed with
an LCQ DECA Ion trap MSn system (Thermo-Finnigan, Belgium)
in the ESI positive mode.
14-Halodaunorubicin (3) via 14-Bromo-13-dimethylketal-
daunorubicin (2). Daunorubicin hydrochloride (1) (16.92 g; 30.0
mmol) was dissolved in a mixture of anhydrous 1,4-dioxane (207
mL) and anhydrous methanol (207 mL). Trimethyl orthoformate
(15.15 mL; 138.5 mmol) was added and the mixture was stirred at
room temperature for 5 min. The solution was cooled to 11 °C and
bromine (2.64 mL; 51.5 mmol) was added over 2 min. The mixture
was stirred at 12 (2 °C for 2 h. Propylene oxide (5.46 mL; 78.0
mmol) was added at 4 °C and the solution was stirred at 2 ( 2 °C
for 75 min. Diisopropyl ether (1.74 L) was added to precipitate
the 14-bromo-13-dimethylketal-daunorubicin intermediate (2). The
red solid was collected by filtration through Whatman no. 42 filter
paper and washed with diisopropyl ether (540 mL).
The solid was dissolved in a mixture of acetone (690 mL) and
0.25 M hydrobromic acid (600 mL). The resulting red solution was
stirred at room temperature for 66 h. HPLC showed that no acetal
remained. The reaction was diluted with water (750 mL) and
extracted with chloroform (3 × 750 mL). Saturated brine (saturated
sodium chloride in water) (150 mL) was added to the aqueous
solution and the product was extracted into n-butanol (2 × 1.5 L;
2 × 0.75 L) with the aqueous layer becoming essentially colorless.
The 1-butanol extract was concentrated under vacuum at 35 °C
(water bath temperature) to a volume of ca. 300 mL. n-Hexane (2
L) was added to precipitate the product, which was collected by
filtration. The resulting red solid was vacuum-dried at room
temperature. LC-MS analysis indicated that the product was a
mixture of the salts of 14-bromodaunorubicin and 14-chlorodauno-
rubicin (3) (ca. 1:1). Thus, 15.46 g of product (3) was isolated [71%
yield from daunorubicin hydrochloride (1), HPLC purity: 96%].
1H NMR (250 MHz, CD3OD) 1.29 (d, 3H, H6′, J ) 6.5 Hz); 1.88
(dd, 1H, H2′, J ) 3.5, 12.7 Hz); 2.04 (ddd, 1H, H2′, J ) 3.5, 12.7,
12.6 Hz); 2.19 (dd, 1H, H8, Jgem ) 4.7, 14.6 Hz); 2.39 (br d, 1H,
H8, Jgem ) 14.6 Hz); 2.78 (d, 1H, H10, Jgem ) 18.6 Hz); 3.05 (d,
1H, H10, Jgem ) 18.6 Hz); 3.57 (m, 1H, H3′); 3.71 (br s, 1H, H4′);
4.01 (s, 3H, H15); 4.29 (br d, 1H, H5′, J ) 6.5 Hz); 4.54 (d, 1H,
H14, Jgem ) 18.0 Hz); 4.67 (d, 1H, H14, Jgem ) 18.0 Hz); 5.01 (s,
1H, H7); 5.47 (d, 1H, H1′, J ) 3.5 Hz); 7.52 (d, 1H, H3, J ) 7.7
Hz); 7.77 (br t, 2H, H1 and H2, J ) 7.7 Hz). m/z (ESI): 562, 564
[M + H]+ for 14-chlorodaunorubicin, 606, 608 [M + H]+ for 14-
bromodaunorubicin.
Sodium 4-Maleimidobutyrate (5). Sodium hydrogen carbonate
(8.391 g; 0.10 mol) was dissolved in water in a 1-L volumetric
flask to produce a 0.1 M solution. A portion of this solution (435
mL; 43.5 mmol) was added slowly via a dropping funnel to a stirred
suspension of 4-maleimidobutyric acid (4) (8.014 g; 43.75 mmol)
in water (80 mL). The resulting solution was stirred for 20 min
and the water was evaporated in vacuo at 30 °C (water bath
temperature) before final drying on a freeze-drying unit. The product

Vectocell-Doxorubicin Therapeutic EValuation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6915
Peptide-Doxorubicin Thioether Conjugate (9b,c). General
procedure: 14-Halodaunorubicin (3) (0.350 mmol) was dissolved
in dry methanol (12 mL) in a round-bottom flask and peptide (7b,c)
(0.85 equiv with peptide content taken into account) was added,
followed by K2CO3 (1.3 equiv) (pH must reach 10; if not, potassium
carbonate was added). The reaction mixture was stirred for 30-90
min (depending on the peptide) under argon and protected from
light. Workup was initiated by the addition of 0.5 M Tris-HCl buffer
pH 9 (1/10 of methanol volume) and extractions with chloroform (6
× 1 volume) until the organic layer became colorless. The aqueous
layer was then loaded on a YMC ODS-A solid-phase extraction
resin (5 g/100 mg of crude compound) preconditioned with
methanol and water in a glass frit. After washes with 0.1% TFA in
water, the conjugate was recovered by elution with methanol.
Methanol was evaporated, the residue was dissolved in water, and
the resulting solution was lyophilized to yield the crude thioether
conjugate. After preparative HPLC purification, pure conjugates
9b,c were isolated. Compound 9b: 78% yield; m/z (ESI) 1111 (M
+ 2H)2+, 2221 (M + H)+; 98.6% purity (surface area, HPLC).
Compound 9c: 84% yield; m/z (ESI) 1318 (M + 2H)2+, 2636 (M
+ H)+; 95.1% purity (surface area, HPLC).
after antimycoplasma treatment that led to a change in its behavior;27
gift of Professor M. M. Mareel, University of Ghent, Belgium),
MCF7/Adr (breast cancer derived from MCF7 parental cell line
by selection with doxorubicin, cultured in the presence of doxo-
rubicin; gift from Professor J. Robert, Institut Bergonie´, Bordeaux,
France), MES-SA/dx5 (human uterine sarcoma, ATCC no.
CRL1977), MDA-MB-231 (human mammary gland adenocarci-
noma, ATCC no. HTB-26). For cytotoxicity studies, viability assays
were performed with a WST-1 cytotoxicity assay (Roche, Man-
nheim, Germany). Briefly, cells were seeded in 96-well plates and
incubated with increasing concentrations of drugs for 1 h. Short-
term exposure to the conjugates was used to prevent any differences
associated with linker stability [release of free doxorubicin (10)]
interfering with the results. Drugs were then washed out and cells
were postincubated with fresh medium for 48 h. The WST-1 test
was performed and IC50 values (drug concentration that inhibits
50% of cell viability) were estimated from sigmoidal regressions
with Graph Pad Prism 3.02 software.
For the in vitro studies with Verapamil (Vp), cells were
preincubated overnight in the presence of 20 µM Vp and then
incubated with same concentration of Vp for the duration of the
experiment. The viability assay was performed as above.
Mice and Tumor Models. Female NMRI nu/nu mice, 6 weeks
of age, were used for in vivo experiments. Mice were monitored
for 1 week in the animal unit prior to experimentation. They
received proper care and maintenance in accordance with institu-
tional guidelines. On day 0, 1 × 107 HCT116 or HCT15 cells
suspended in 100 µL of Dulbecco’s modified Eagle medium
(DMEM) (Invitrogen, France) or 3 × 106 MDA-MB-231 cells
suspended in 100 µL of matrigel/DMEM (1/2 v/v) (BD Biosciences,
France) were injected in the ventral skin of the mice.
Immunostaining to detect Pgp expression was performed on 10
µm thick tumor sections with a rabbit anti-human mdr1 antibody
(Dako, France), according to the manufacturer’s recommendations,
and a secondary goat anti-rabbit antibody (Dako, France) and a
DAB substrate (Dako, France).
Chemotherapy treatment began 3 days (HCT116 and HCT15)
or 7 days (MDA-MB-231) after tumor implantation, when tumors
reached a minimal size of 70 mm3. Doxorubicin (10) and DPV1047-
E-Dox (8a) were dissolved in NaCl 0.9%/H2O (9/1 v/v) and filtered
through a 0.2 µm filter, to obtain a concentration corresponding to
the required dose to be administered in a volume of 10 µL/g of
body weight. Drug concentrations were controlled with a spectro-
photometer (U-2000, Hitachi). Drugs were administered following
a (Q2D3) × 3W schedule, that is, three injections every 2 days
during 3 weeks (nine injections in total). The cumulative doses of
each compound were 54 µmol/kg for doxorubicin (10) and 72 or
90 µmol/kg for DPV1047-E-Dox (8a).
N-Maleimidobutyrate Doxorubicin (11). Doxorubicin hydro-
chloride (10) (0.05 g, 0.086 mmol, 1 equiv) and 4-maleimidobutyric
acid (4) (0.015 g, 0.086 mmol, 1 equiv) were dissolved in DMF
(3.5 mL). Diisopropylethylamine (DIPEA) (0.03 mL, 0.172 mmol,
2 equiv) was added and the solution was stirred for 15 min at room
temperature. Then, HATU (0.036 g, 1.1 equiv) previously dissolved
in DMF (1 mL) was added and the mixture was stirred for an
additional hour at room temperature. The solution was evaporated
at reduced pressure (water bath at 30 °C). The red residue was
dissolved in water and purified on reverse-phase silica (YMC). The
column was eluted with a stepwise gradient of acetonitrile (10%,
20%, 25%, 30%, and 50%) in water and the fractions were checked
by HPLC. The product fractions were combined and the solvent
was removed, initially in a rotary evaporator bath (at 30-32 °C),
and then on a freeze-drying unit. The product (11) was obtained as
1
a red solid (0.044 g; 71% yield). H NMR (400 MHz, DMSO-d6)
1.31 (d, 3H, H6′, J ) 6.4 Hz); 1.41 (dd, 1H, H4′, J ) 4.4, 12 Hz);
1.63 (m, 2H, H3′′); 1.83 (dt, 1H, H3′, J ) 4, 12.8 Hz); 2.01 (t, 2H,
H2′′, J ) 6 Hz); 2.08 (dd, 1H, H2′, J ) 5.6, 14 Hz); 2.18 (m, 1H,
H2′); 2.64 (br d, 1H, H8, J ) 15.8 Hz); 2.73 (m, 1H, H8); 2.95
(m, 2H, H4′′); 3.42 (m, 1H, H10); 3.92 (m, 1H, H10); 3.94 (s, 3H,
H15); 4.15 (m, 1H, H5′); 4.57 (m, 2H, H14); 4.71 (m, 2H, H7);
5.20 (m, 1H, H1′); 6.94 (s, 2H, H6′′); 7.51 (d, 1H, H3, J ) 8 Hz);
7.59 (m, 1H, NH); 7.86 (m, 2H, H1 and H2). m/z (ESI): 709 (M
+ H)+; 86.1% purity (surface area, HPLC).
Peptide-Doxorubicin Amide Conjugate (12a,b). General
procedure: A solution of N-maleimidobutyrate doxorubicin (11)
(0.025 g, 0.035 mmol, 1 equiv) in DMF (0.7 mL) was added at
room temperature under argon to a solution of peptide (7a,b) (0.7
equiv with peptide content taken into account) in oxygen-free water
(7 mL). The resulting solution was stirred at room temperature under
argon for 2 h and then extracted three times with dichloromethane.
The aqueous layer was lyophilized to give the crude peptide
conjugate as a red solid. The solid was purified by means of
preparative HPLC separation. The relevant fractions (checked by
HPLC) were combined and the resulting solution was lyophilized
to give doxorubicin-peptide conjugate (12a,b) TFA salt as a red
Evaluation of Antitumoral Activity. Tumor volumes were
calculated by the following formula: length (millimeters) × width2
(square millimeters)/2. Results are expressed as mean T/C, that is,
mean tumor volume of treated versus control mice. Statistical
analyses are performed with GraphPad Prism program. A one-way
ANOVA was assessed, followed by a Newman-Keuls test, to
compare the antitumor activity of the Vectocell-doxorubicin
conjugate [DPV1047-E-Dox (8a)] to that of doxorubicin. *p e
0.05, **p e 0.01, ***p e 0.001.
Acknowledgment. We gratefully acknowledge advice and
suggestions given by Dr. John Tchelingerian, Dr. Denis Ravel,
Dr. Catherine de Coupade, and Xavier Ranc¸on. We also thank
Drs. Thierry Huet, Ce´cile Bougeret, and Marc Hillairet de
Boisferon for support with these experiments. This work was
supported in part by the Agence Nationale pour la Valorisation
de la Recherche (ANVAR, France). Vectocell is a trademark
of Diatos SA. All rights are reserved.
solid. Compound 12a: 34% yield; m/z (ESI) 1565 (M + 2H)2+
;
95.9% purity (surface area, HPLC). Compound 12b: 97% yield;
m/z (ESI) 802 (M + 3H)3+, 1202 (M + 2H)2+, 2405 (M + H)+;
93.7% purity (surface area, HPLC).
All the tested compounds are produced as TFA salts. Previous
experiments performed with DPV-maleimide conjugates, which
were synthesized as TFA salts, in vitro and in vivo2 showed that
the use of TFA for synthesis is not associated with toxicity up to
1 mM concentration.
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