Design, synthesis and preliminary biological evaluations of novel amphiphilic drug carriers

Article abstract of DOI:10.1016/j.bmcl.2005.11.107

The synthesis of a new fluorocarbon amphiphilic drug carrier is described. A polyfunctional amino acid endowed with a fluorocarbon chain and a sugar moiety providing the amphiphilic character constitutes the central element of this structure. A ¹⁴

Full text of DOI:10.1016/j.bmcl.2005.11.107

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1111–1114
Design, synthesis and preliminary biological evaluations
of novel amphiphilic drug carriers
a
a,
a
b
*
Sandrine P e´ rino, Christiane Contino-P e´ pin, Sylvain Jasseron, Maryse Rapp,
b
a,
*
Jean-Claude Maurizis and Bernard Pucci
a
Laboratoire de Chimie Bioorganique et des Syst e` mes Mol e´ culaires Vectoriels, Facult e´ des Sciences,
3, rue Louis Pasteur, 84000 Avignon, France
3
INSERM U484, rue Montalembert, BP 184, 63005 Clermont-Ferrand Cedex, France
b
Received 15 November 2005; revised 25 November 2005; accepted 28 November 2005
Available online 4 January 2006
Abstract—The synthesis of a new fluorocarbon amphiphilic drug carrier is described. A polyfunctional amino acid endowed with a
1
4
fluorocarbon chain and a sugar moiety providing the amphiphilic character constitutes the central element of this structure. A C-
radiolabelled acetyl group was grafted onto the third function and the bioavailability of this molecule was specified in mice after IV
administration. This amphiphilic drug carrier exhibits a rapid and homogeneous distribution to the whole tissues and slow elimi-
nation half-lives (higher than one day) through a biliary excretion without any toxicity (no measured DL 50 for concentrations
up to 500 mg/kg).
ꢀ
2005 Elsevier Ltd. All rights reserved.
The preparation of drug bearing macromolecular conju-
–5
gates has been widely explored since the last decade.
To this purpose, we focused our investigations on the
design of amphiphilic multifunctional molecular carriers
with a modular structure including an amino acid scaf-
fold upon which are anchored several architectural
components:
1
These systems exhibited therapeutic molecules linked
to a polymeric backbone by means of biodegradable
covalent bonds. In this field, we have developed
researches on the biomedical potentialities of a new class
6
of oligomeric carriers called ‘‘Telomers’’. Preliminary
biological studies have shown that these multifunctional
macromolecules exhibit good biocompatibility making
• A perfluoroalkyl tail as a hydrophobic part.
• A glycosidic or peptidic hydrophilic head providing
the solubility in water and/or a cell targeting.
• A labelling domain for the in vivo follow-up and/or a
drug moiety.
7
,8
them suitable for drug delivery systems. Therefore,
the grafting of cytotoxic agents and possibly different
targeting devices to the polymeric backbone of telomers
resulted in macromolecular prodrugs with an increased
bioavailability and a better therapeutic index than the
The general structure of the investigated molecules is
shown in Figure 1. We can expect that, compared to tel-
omers, the combination of these structural domains on a
molecular carrier should provide less complicate data
interpretation with more wide therapeutic exploitation.
As regards the hydrophobic tail, it has to be specified
6
,9
parent drugs.
Nevertheless, despite the numerous
therapeutic potentialities exhibited by our so-called ‘‘tel-
omers’’, the several difficulties in predicting and control-
ling the drug-telomer conjugate architecture prompted
us to develop a new class of multivalent carriers, having
the main potentialities and advantages of telomers but
with a well-defined structure.
glycosidic or peptidic hydrophilic HEAD
perfluoroalky
amino acid CORE
hydrophobic TAIL
Keywords: Drug carrier; Amphiphilic carrier; Radiolabelling;
Bioavailability.
*
Corresponding authors. Tel.: +33490144442; fax: +33490144449;
e-mail addresses: christine.contino@univ-avignon.fr; bernard.pucci@
univ-avignon.fr
labeling PROBE and/or DRUG
Figure 1. General structure of new amphiphilic molecular carriers.
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.107

1112
S. P e´ rino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1111–1114
that a fluoroalkyl chain was chosen instead of a classical
hydrocarbon one to minimize the cell toxicity of such
amphiphilic molecules. Indeed, it is well known that
hydrocarbon and fluorocarbon chains exhibit poor mis-
cibility, thus fluorocarbon surfactants and phospholip-
compound 4. Its nonradiolabelled analogue 4a was syn-
thesized likewise from acetic anhydride. This molecule
4a was fully characterized by NMR spectroscopy and
mass spectra. The specific radioactivity of the C-la-
belled derivative 4 was 1.34 lCi/mg.
1
3
14
1
0
ids of biological membranes do not mix very well. In
those conditions, fluorocarbon amphiphiles do not
destabilize cell membranes at a concentration higher
than their critical micellar concentration (CMC) and
According to the chemical structure of the compound 4,
we predicted that it would exhibit amphiphilic behav-
iour in biological fluids. For this purpose, we focussed
our attention on the determination of its essential ten-
sioactive properties. All these physicochemical studies
were performed on the nonradiolabelled analogue 4a.
1
1
are less toxic than hydrocarbon analogues.
In this context, we preliminary investigated the in vivo
behaviour and biocompatibility of the single carrier
through the follow-up of a radiolabelled derivative.
We describe herein the synthesis and biodistribution in
The surface tension vs concentration of its aqueous solu-
tion was measured at 20 ꢁC by using a KRUSS R12 ten-
siometer, following the Wilhelmy plate method. The
critical aggregation concentration (CAC) value of this
1
4
mouse of amphiphilic carrier 4, labelled with C.
À2
The synthesis of the radiolabelled molecule 4 was per-
formed easily as summarized in Figure 2.
compound was 1.61 · 10 mM. Such a low value could
be ascribed to the presence of the fluorocarbon chain
which generally induces a dramatic decrease of the
CMC (or CAC) by comparison with hydrocarbon
After reduction of the azide derivative 1, previously
obtained from the corresponding commercial iodide, a
suitable lysinoyl protected moiety was introduced on
the molecular architecture using conventional methods
of peptidic synthesis. The consecutive removal of the
Boc N-protecting group of lysine was followed by cou-
pling lactobionolactone achieved from dehydration of
lactobionic acid. The acylation of the sugar hydroxyl
groups, performed by using acetic anhydride in pyridine,
1
4
analogues.
Moreover, the tensiometric curve led us to specify the
polar head interfacial cross-section of compound 4a by
2
˚
applying the Gibbs absorption isotherm: a = 56 A
(25 ꢁC). This value is in good agreement with measure-
ments already performed on various hydrocarbon or
fluorocarbon surfactants derived from lactobionamide
1
2
2
15
˚
(a = 40–50 A ).
gave compound 3 in satisfactory yield. Then, after
hydrogenolysis of the benzyloxycarbonyl protective
function of the lysine e-amino group, we have acylated
The particles size that compound 4a formed in water at
a concentration higher than its CAC was observed at
25 ꢁC by light scattering with a Zetasizer Nano-S model
ZEN 1600 granulometer.
1
4
the resulting amine moiety and introduced a C atom
by using C-labelled acetic anhydride in pyridine.
1
4
Finally, a deacetylation of the saccharidic moiety by
transesterification in methanol with catalytic amount
of sodium methylate was performed to provide
This measurement led us to specify the mean hydrody-
namic diameter of these particles: 26 nm (average size
measured with six different batches) whatever the con-
centration used between 0.05 and 0.5 mM (Fig. 3). Such
a value was markedly higher than that usually measured
for micelles and could be ascribed to aggregates. Thus,
amphiphilic compound 4a, which displays a very good
solubility in water, self-organizes at low concentration
as supramolecular aggregates different from classic
micellar systems whose average size is usually lower
than 5 nm. Such a behaviour is frequently observed with
fluorocarbon surfactants which have a strong tendency
O
BocHN
8 17
C F
,
c
N
H
C
8
F
17
8 17
C F
I
N
3
1
2
NH(Z)
OAc
O
AcOOAc
AcO
OAc O
OAc
H
N
d, e, f
8 17
C F
O
N
H
1
6,17
O
to form cylindrical or rod-like micelles.
OAc
OAc
3
NH(Z)
OH
OH
O
HOOH
HO
O
H
N
g, h, i
OH
C8F17
O
N
H
O
OH
OH
4
carbone 14
*
(
4a : non radiolabelled analogue
)
NH
*
O
1
4
Figure 2. Synthesis of the C-labelled carrier. Reagents: (a) NaN
DMF (85%); (b) H /Pd/C (100%); (c) BocLys(Z)OH, DCC, HOBT
Cl (100%); (e) lactobionic acid, TEA, MeOH; (f)
O, Py (65%); (g) H
3
,
2
(
79%); (d) TFA/CH
AcO)
2
2
Figure 3. Histogram aggregate size distribution of compound 4a in
aqueous solution determined as hydrodynamic diameter versus volume
percentage of populations by DLS.
14
(
2
2
/Pd/C (100%); (h) C-labelled acetic
À
+
anhydride, Py (46%); (i) MeO Na /MeOH (84%).

S. P e´ rino et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1111–1114
1113
Table 1. Distribution of radioactivity in tissues after iv administration of 13.4 lCi of labelled carrier
1
5 min
1 h
6 h
24 h
9.47 ± 0.88
T1/2 (h)
Blood
Kidney
Liver
31.97 ± 2.97
13.49 ± 3.02
15.84 ± 5.76
12.73 ± 2.50
27.05 ± 4.09
9.21 ± 1.92
10.95 ± 1.95
1.03 ± 0.2
22.85 ± 1.78
15.29 ± 0.94
16.32 ± 1.82
11.22 ± 2.42
22.64 ± 1.95
10.92 ± 3.25
10.82 ± 2.36
0.77 ± 0.17
13.31 ± 0.58
9.62 ± 1.33
32.5
48.2
9.04 ± 1.01
31.77 ± 3.27
5.42 ± 1.11
9.97 ± 1.72
7.63 ± 0.67
8.94 ± 2.78
0.49 ± 0.07
11.49 ± 1.19
8.94 ± 1.22
25.97 ± 2.08
4.73 ± 0.49
13.21 ± 2.27
6.44 ± 1.00
6.53 ± 1.38
0.38 ± 0.04
6.81 ± 1.45
138.4
38.2
ND
Heart
Lung
Spleen
Skin
53.7
42.7
72.6
36.8
Brain
Tumour
5.08 ± 1.31
Results, given in percentage of administered dose per g of tissue, are means of five measures ± SD.
Male C57B16 mice were grafted by subcutaneous injec-
tion of 10 syngenic B16 melanoma cells. Animals were
• First, this amphiphilic fluorinated carrier exhibits a
half-life time higher than one day in all tissues and
in particular in blood. This result suggests that the
binding of an active principle to such a carrier
could permit to extend the presence of the drug
into the whole organism, including tumoural tis-
sues, and to increase its pharmacological effect.
Moreover, several biological studies showed that
compound 4 did not exhibit any lethal toxicity up
to a concentration of 500 mg/kg after IV adminis-
tration in mice (data not shown). Thus, these preli-
minary biological findings appear to substantiate
the possible use of these new amphiphilic carriers
for delayed delivery of drugs.
6
used when the mean diameter of the tumours reached
0
.5 cm. 10 mg of the labelled compound 4 (13.4 lCi) dis-
solved in 200 ll of physiological serum was injected in
the caudal vein. Animals were sacrificed by CO inhala-
tion 15 min, 1, 6 and 24 h after administration. Mice
were immediately frozen in liquid nitrogen and sliced
2
(
40 lm) using a cryomicrotome. Tissue radioactive con-
centration was determined with a computer-controlled
multiwire proportional counter (Ambis 4000) allowing
1
8
direct quantitation of regions of interest. Elimination
half-lives were determined using the Kinetica 2000 soft-
ware (Innaphase).
•
Second, the high concentration of radioactivity mea-
sured within the lungs can be reasonably ascribed
to the C-CO arising from the catabolism of the
e-acetyl group of lysine. Such a result has to be
favourably considered, indeed it suggests that a drug
linked to this amphiphilic carrier through an amide
group may be released over a long period within the
organism.
The radioactivity follow-up performed on C57B16 mice
bearing grafted B16 syngeneic melanoma tumours
showed a rapid and relatively homogeneous distribution
of the radiolabelled molecule to the whole tissues, except
brain (Table 1). Maximal tissue concentrations were ob-
served 1 h after administration. Following this distribu-
tion phase, slow elimination half-lives were measured. In
the blood, this one was 32 h, while in the other tissues
such as tumour or spleen, longer half-lives were deter-
mined (37 h for the tumour, 54 h for the spleen).
1
4
2
• However, the ubiquitous distribution of the carrier
and above all its great affinity for the liver could pos-
sibly induce an increase of the whole toxicity of the
drug. Thus, to overcome such a drawback, it would
be attractive to endow this molecular amphiphilic
carrier with specific moieties providing a selective
affinity for tumoural sites. In this field, we previously
directed chemotherapeutic agents to highly vascular-
ized zones surrounding tumours by grafting RGD
sequences onto the polymeric backbone of our
High radioactive concentrations in the liver after 6 h
indicate an important biliary excretion. The elimination
half-life was the highest (138 h) in this tissue. Two
hypotheses can be proposed to explain such a behav-
iour. We might assume that the size of the particles
formed in water by the carrier 4 induces its partial accu-
mulation within the liver as it is usually observed for
9
THAM-derived telomers. The application of such a
1
9,20
various liposomes or nanoparticles.
cell targeting concept to our amphiphilic multifunc-
tional molecular carriers is currently undertaken. In
those compounds, a peptidic homing device will con-
stitute the hydrophilic head of the carrier.
More probably, considering the small size (26 nm) of the
aggregates produced by compound 4, these accumula-
tion and retention times could be the consequence of a
chemical interaction between the galactopyranosyl moi-
ety of compound 4 and specific receptors present in the
liver. Indeed, b-galactose residues are known to be rec-
ognized by asialoglycoproteins receptors (ASGP-R)
In conclusion, all the physicochemical and biological
studies carried out with the fluorinated amphiphilic
compound 4 seem to validate its therapeutic potentialities
as a promising vector for targeted drug delivery to tu-
mours as well for molecular imaging of neoangiogenesis.
2
1
present on the surface of hepatocytes. Also, several
lactose-endowed macromolecules devoted to promoting
an efficient entry in liver cells are described in the
2
2,23
literature.
References and notes
These results warrant several remarks and comments on
the potentialities of such an amphiphilic carrier:
1. Nori, A.; Kopecek, J. Adv. Drug Delivery Rev. 2005, 57,
609.

1114
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2
3
. Kopecek, J.; Kopecekova, P.; Minko, T.; Lu, Z. R. Eur. J.
Pharm. Biopharm. 2000, 50, 61.
. Duncan, R.; Gac-Breton, S.; Keane, R.; Musila, R.; Sat,
Y. N.; Satchi, R.; Searle, F. J. Controlled Release 2001, 74,
13. Compound 4a: white powder (20%), mp 145.6–148.9 ꢁC
(dec); [a ]: + 6 (C, 1, MeOH); MS (ESI) m/z: 974.5
(M+H) ; 996.5 (M+Na) ; H NMR (250 MHz, DMSO-
D
+
+ 1
6
d , d): 8.12 (t, 1H), 7.71 (t, 1H), 7.22 (t, 1H), 5.47 (d, 1H),
1
35.
5.19 (d, 1H), 4.82 (m, 2H), 4.64–4.51 (m, 3H), 4.36–4.18
(m, 3H), 3.73–3.35 (m, 5H), 2.96 (m, 2H), 2.47–2.39 (m,
4
5
6
. Ouchi, T.; Ohya, Y. Prog. Polym. Sci. 1995, 20, 211.
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. Contino-Pepin, C.; Maurizis, J. C.; Pucci, B. Curr. Med.
Chem. 2002, 2, 645.
. Zarif, L.; Riess, J. G.; Pucci, B.; Pavia, A. A. Biomat. Art.
Cells & Immob. Biotech. 1993, 21, 597.
. Maurizis, J. C.; Azim, M.; Rapp, M.; Pucci, B.; Pavia, A.;
Madelmont, J. C.; Veyre, A. Xenobiotica 1994, 24, 535.
. Jasseron, S.; Contino-Pepin, C.; Maurizis, J. C.; Ollier,
M.; Pucci, B. Eur. J. Med. Chem. 2003, 38, 825.
1
9
2H), 1.99 (s, 3H), 1.59–1.23 (m, 6H). F NMR (250 MHz,
DMSO-d
, d): À80.22 (s, 3F), À113.45 (s, 2F), À121.7 (s,
6
1
3
6F), À122.5 (s, 2F), À123.2 (s, 2F), À125.76 (s, 2F).
C
7
8
9
NMR (250 MHz, DMSO-d , d): 172.9; 170.0; 169.1; 105.3;
6
83.9; 76.3; 73.7; 72.5; 71.8; 71.6; 68.6; 62.7; 61.0; 52.6; 48.0;
44.6; 42.9; 41.0; 32.0; 27.6; 23.1; 21.7.
14. Kissa, E. In Fluorinated Surfactants: Synthesis, Properties,
Application; Surfactants Science Series 50; Dekker: New
York, 1994; Chapter 6–7, pp 191–320.
1
1
1
0. Barth e´ l e´ my, P.; Tomao, V.; Selb, J.; Chaudier, Y.; Pucci,
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Veyre, A. Eur. J. Nucl. Med. 1998, 25, 109.
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2. Compound 3: white powder (51%), mp 65 ꢁC (dec); [a
D
]: +
(C, 1, CHCl ); MS (ESI) m/z: 1403 (M+H) ; 1425
+
3
3
+
1
(
M+Na) ; H NMR (250 MHz, DMSO-d , d): 8.07 (m,
6
2
2
2
1
H), 7.34 (s, 5H), 7.01 (m, 1H), 5.47 (d, 1H), 5.30–5.10 (m,
H), 5.02–4.79 (m, 5H), 4.51–3.90 (m, 8H), 3.35 (m, 2H),
.96 (m, 2H), 2.30 (m, 2H), 2.14, 2.09, 2.04, 2.01, 1.99,
1
9
.96, 1.92 (s, 27H), 1.59–1.23 (m, 6H).
F NMR
(
250 MHz, DMSO-d
6
, d) : À80.22 (s, 3F), À113 (s, 2F),
À121.4 (s, 6F), À122.2 (s, 2F), À123 (s, 2F), À125.4 (s,
1
3
2
1
1
6
2
F). C NMR (250 MHz, CDCl , d): 171.3; 170.5; 170.1;
3
70; 169.7; 169.2; 167.9; 156.7; 136.7; 128.4; 128; 127.9;
01.6; 77.9; 72.7; 71.1; 70.9; 70; 69.4; 69; 66.9; 66.5; 61.6;
1; 52.6; 40.4; 31.9; 31.2; 30.5; 29.1; 22.2; 20.6; 20.5; 20.4;
0.3.