Preparation and characterization of novel poly(ethylene glycol) paclitaxel derivatives

Article abstract of DOI:10.1016/j.ijpharm.2013.05.027

Paclitaxel has been found to be very effective against several human cancers; one of the major problems with its use is its poor solubility, which makes necessary its solubilization with excipients that can determine allergic reactions often severe. The a

Full text of DOI:10.1016/j.ijpharm.2013.05.027

International Journal of Pharmaceutics 454 (2013) 653–659
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Preparation and characterization of novel poly(ethylene glycol)
paclitaxel derivatives
a,∗
a
b
a
Silvia Arpicco a , Barbara Stella , Oddone Schiavon , Paola Milla ,
Daniele Zonari , Luigi Cattela
Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Via Pietro Giuria 9, 10125 Torino, Italy
a
b
Dipartimento di Scienze Farmaceutiche, University of Padova, Via Francesco Marzolo 5, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Paclitaxel has been found to be very effective against several human cancers; one of the major problems
with its use is its poor solubility, which makes necessary its solubilization with excipients that can deter-
mine allergic reactions often severe. The aim of this study is to develop highly water-soluble prodrugs
of paclitaxel. For this purpose we prepared a series of new paclitaxel–poly(ethylene glycol) (PEG) conju-
gates that were characterized and evaluated for their in vitro stability and cytotoxicity. In particular, in
order to modulate the release of paclitaxel from prodrugs, we prepared different compounds introducing
Received 15 January 2013
Received in revised form 10 May 2013
Accepted 13 May 2013
Available online 20 May 2013
Keywords:
Paclitaxel
Poly(ethylene glycol)
Prodrugs
ꢀ
PEG in the drug C2 and/or C7 positions via ester or carbamate linkage. The conjugates were obtained
in high purity and good yield. The carbamate prodrugs were highly stable in different media, while the
ꢀ
compounds obtained linking PEG at C2 position through an ester bond showed lower stability. Finally,
Stability
the cytotoxic activity of the conjugates was evaluated on two cancer cell lines and the results showed
that all the derivatives had a reduced cytotoxicity compared to that of paclitaxel.
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
1994; Fjallskog et al., 1993), and premedication with corticoste-
roids and antihistamines is often required (Weiss et al., 1990). In
order to overcome these problems, new aqueous-based formula-
Paclitaxel (PTX) is a natural product isolated from the bark of
®
Taxus breviflolia (Pacific yew tree) and is today considered to be one
of the most important drugs in cancer chemotherapy for the clin-
ical treatment of many types of cancers (Kingston and Newman,
tions for PTX, that do not require solubilization by Cremophor EL ,
have been developed (Marupudi et al., 2007; Skwarczynski et al.,
2006). The prodrug strategy is a promising way in terms of improv-
ing the drug solubility and keeping the pharmacological functions
unaltered (Stella and Nti-Addae, 2007). Several reports of water-
soluble prodrugs of PTX have been reported that are considered
to improve water solubility of the parent drug and to avoid the
use of toxic detergents during administration (Vyas and Vittorio,
1995).
2
007; Rowinsky, 1997). It is active against a number of cancer
types including breast, lung, prostate, ovarian and some leukaemias
Bonomi et al., 1997; Bookman et al., 1996; Nabholtz et al., 1996;
(
Wani et al., 1971). At molecular level, PTX exerts its antitumor
activity by interacting with tubulin (Schiff et al., 1979). In con-
trast to other anti-mitotic agents, such as Vinca alkaloids, which
act to inhibit microtubule formation, PTX promotes tubulin poly-
merization and stabilizes the microtubules. Therefore, cell division
is blocked in the late G2 mitotic phase of cell cycle (Kumar, 1981;
Manfredi and Horwitz, 1984). However, limited response rates
and significant side effects are the major obstacles for more effec-
tive cancer therapy. Additionally, PTX’s very low water solubility
is a real problem in intravenous administration; PTX is currently
One of the most used polymers for prodrug delivery is
poly(ethylene glycol) (PEG) (Greenwald et al., 2003). PEG is an
amphiphilic polymer that is soluble in organic solvents as well as
in water, non-toxic and is eliminated from the body by a combi-
nation of renal and hepatic pathways; thus, this molecule is ideal
to be employed in pharmaceutical applications; moreover, PEG has
been approved by the FDA for human intravenous, oral and der-
mal applications (Hooftman et al., 1996). Some papers describe the
different synthetic approaches adopted to covalently attach PEG
®
administered in a vehicle containing Cremophor EL (polyethoxy-
lated castor oil) and ethanol. Significant side effects associated
with hypersensitivity to Cremophor EL have been observed (Dorr,
®
ꢀ
to PTX: PEG of various molecular weights was linked to the C2
and/or C7 positions of PTX through different bonds either directly
or through suitable spacers (i.e. amino acids) or linkers (Feng et al.,
2002; Greenwald et al., 1994, 1996; Li et al., 1996; Schoenmakers
et al., 2004).
∗ Corresponding author. Tel.: +39 011 6706668; fax: +39 011 6706663.
E-mail address: silvia.arpicco@unito.it (S. Arpicco).
0
378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.05.027

6
54
S. Arpicco et al. / International Journal of Pharmaceutics 454 (2013) 653–659
However, there are still some problems related to PEG–PTX pro-
3.34 (d, 1H, C3-H), 4.17 and 4.28 (d, 2H, C20-H), 4.48 (dd, 1H, C7-H),
ꢀ
drugs described in literature till now, such as for example poor
stability or low improvement of solubility that lead to a limitation
in their clinical use (Skwarczynski et al., 2006). Thus, a very attrac-
tive challenge in this field is still to obtain new stable water-soluble
PTX conjugates with improved activity.
4.96 (d, 1H, C5-H), 5.51 (d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 5.80 (d,
ꢀ
1H, C3 -H), 6.21 (t, 1H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH),
ꢀ
ꢀ
ꢀ
7.3 (m, 3 -Ph), 7.4 (m, 3 -NBz), 7.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1
(d, 2-OBz).
ꢀ
The
carboxyl
function
of
2 -succinyl-paclitaxel
(1)
To reach this goal, the aim of this study was to develop highly
water-soluble prodrugs of PTX. For this purpose, we prepared a
series of new PEG–PTX conjugates that were characterized and
evaluated for their in vitro stability and cytotoxicity. In particu-
lar, in order to modulate the release of paclitaxel from prodrugs,
(20 mg, 0.0210 mmol) was activated in the corresponding
N-hydroxysuccinimidil derivative (2) by reaction with N-
hydroxysuccinimide (NHS) (3.2 mg, 0.0278 mmol) in the presence
ꢀ
of N,N -dicyclohexylcarbodiimide (DCC) (5.6 mg, 0.0269 mmol)
in dry dichloromethane. The reaction mixture was stirred for 6 h
at room temperature. After filtration and evaporation the crude
product was dissolved in dichloromethane and washed with brine
and did not required any further purification step (19 mg, yield
ꢀ
we prepared several conjugates introducing PEG in the drug C2
and/or C7 positions using different synthetic routes and the proper-
ties of these derivatives are discussed, together with a preliminary
examination of their in vitro antitumor activity.
85%). 1H NMR (CDCl ): ı 1.11 (s, 3H, C17-H), 1.19 (s, 3H, C16-H),
3
1
3
.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H), 2.2 (m, 2H, C14-H), 2.23 (s,
ꢀ
H, C10-OAc), 2.43 (s, 3H, C4-OAc), 2.63 (m, 2H, 2 -OCOCH ), 2.66
2
2
. Materials and methods
(
m, 4H, NCOCH CH CO), 2.92 (m, 2H, CH CON), 3.34 (d, 1H, C3-H),
2 2 2
4
5
1
.17 and 4.28 (d, 2H, C20-H), 4.48 (dd, 1H, C7-H), 4.96 (d, 1H, C5-H),
2
.1. Materials and instruments
ꢀ
ꢀ
.51 (d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 5.80 (d, 1H, C3 -H), 6.21 (t,
ꢀ
H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph), 7.4
Unless stated otherwise, all reagents and solvents were obtained
ꢀ
ꢀ
(m, 3 -NBz), 7.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
from commercial sources and were used without further purifica-
tion. Paclitaxel was a gift from Indena (Milan, Italy). PEG derivatives
2
.2.2. Preparation of 4-nitrophenyl-carbonate paclitaxel
derivatives
The different carbonate paclitaxel derivatives (5–7) were pre-
(
5
alpha-methoxy-omega-amino poly(ethylene glycol), m-PEG-NH2
and 20 kDa) were purchased from IRIS Biotech GmbH (Marktred-
witz, Germany).
pared following the method described by de Groot (de Groot
et al., 2000) with minor modifications. The reactions were car-
ried out under an argon atmosphere. PTX (50 mg, 0.0585 mmol)
was dissolved in dry dichloromethane containing 4 drops of pyri-
All reactions requiring anhydrous conditions were performed
under an Ar or N2 atmosphere.
The reactions were monitored by thin-layer chromatography
(
TLC) on F254 silica gel pre-coated sheets (Merck, Milan, Italy); after
ꢀ
dine. For the preparation of 2 -(4-nitrophenyl carbonate)paclitaxel
development, the sheets were visualized by irradiation by UV light
and/or by exposition to iodine vapor. Flash-column chromatogra-
phy was performed on 230–400 mesh silica gel (Merck).
(5), 200 mg (1.06 mmol) of 4-nitrophenyl chloroformate in dry
dichloromethane was added and the reaction proceeded for 5 h at
◦
ꢀ
−
35 C. In the case of the synthesis of 2 ,7-(4-nitrophenyl biscar-
bonate)paclitaxel (7) PTX was reacted with 300 mg (1.59 mmol) of
-nitrophenyl chloroformate for 24 h at room temperature. Then
HPLC analyses were carried out using a LiChroCART C18 column
(
250 mm × 4 mm i.d., 5 ␮m particle size) equipped with a C18 col-
4
umn guard (Merck) on a Merck-Hitachi HPLC system. The column
was eluted using two solvents: water with 0.05% trifluoroacetic acid
the reaction mixtures were washed with a solution of potassium
bisulfate and dried with anhydrous magnesium sulfate. The solvent
was then removed under reduced pressure and the crude prod-
ucts were purified by flash chromatography (hexane/ethyl acetate
(
TFA) (solvent A) and acetonitrile with 0.05% TFA (solvent B). The
flow rate was maintained at 1 mL/min using a gradient protocol as
follows: solvent A 90% for 5 min, a linear gradient from A 90% to A
1
A 90% for 5 min. The eluting fractions were monitored at 227 nm
using an L4000UV detector. Peak heights and areas were recorded
and processed on a CBM-10A Shimadzu interface (Shimadzu, Milan,
Italy).
5
5/45 (v/v) for 5, 60/40 (v/v) for 6 and 70/30 (v/v) for 7). 7-(4-
0% for 30 min, A 10% for 10 min, a linear gradient from A 10% to
Nitrophenyl carbonate)paclitaxel (6) was directly obtained starting
from the crude 2 ,7-(4-nitrophenyl biscarbonate)paclitaxel (7) that
was left for one night in the column before purification.
ꢀ
Compound 5, yield 65% (38 mg) 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
2
The 1H nuclear magnetic resonance ( H NMR) spectra were
recorded on a Bruker 300 Ultrashield instrument (Karlsruhe,
Germany) in CDCl3 solution at room temperature, with SiMe4 as
internal standard. UV–vis spectra were obtained on a Beckman 730
spectrophotometer (Beckman Coulter, Milan, Italy).
1
.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.34
(
(
d, 1H, C3-H), 4.17 and 4.28 (d, 2H, C20-H), 4.48 (dd, 1H, C7-H), 4.96
d, 1H, C5-H), 5.51 (d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 6.10 (dd, 1H,
ꢀ
ꢀ
C3 -H), 6.21 (t, 1H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH),
7
7
ꢀ
ꢀ
.3 (m, 3 -Ph), 7.35 (d, nitrophenyl), 7.4 (m, 3 -NBz), 7.5 (m, 2-OBz),
ꢀ
.73 (d, 3 -NBz), 8.1 (d, 2-OBz), 8.26 (d, nitrophenyl).
2
2
.2. Chemistry
Compound 6 yield 74% (44 mg) 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
2.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.34
(d, 1H, C3-H), 4.17 and 4.28 (d, 2H, C20-H), 4.80 (d, 1H, C2 -H), 4.96
ꢀ
.2.1. Preparation of paclitaxel-2 -succinyl-NHS
ꢀ
ꢀ
2
-Succinyl-paclitaxel (1) was prepared as reported else-
where with modifications (Deutsch et al., 1989). Briefly, PTX
33 mg, 0.0386 mmol) was dissolved in 500 ␮L of dry pyridine
to which 7.7 mg of succinic anhydride (0.0772 mmol) and 0.5 mg
0.00386 mmol) of 4-dimethylaminopyridine were added. The
(d, 1H, C5-H), 5.26 (dd, 1H, C7-H), 5.67 (d, 1H, C2-H), 5.78 (d, 1H,
ꢀ
(
C3 -H), 6.21 (t, 1H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH),
ꢀ
ꢀ
7.3 (m, 3 -Ph), 7.35 (d, nitrophenyl), 7.4 (m, 3 -NBz), 7.5 (m, 2-OBz),
ꢀ
(
7.73 (d, 3 -NBz), 8.1 (d, 2-OBz), 8.26 (d, nitrophenyl).
resulting solution was stirred for 3 h at room temperature. The
product was purified by flash chromatography with elution in chlo-
roform/methanol (90/10, v/v) to give 34.6 mg of pure product, 94%
Compound 7 yield 86% (59 mg) 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
2.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.34
(d, 1H, C3-H), 4.17 and 4.28 (d, 2H, C20-H), 4.96 (d, 1H, C5-H), 5.28
yield. 1H NMR (CDCl ): ı 1.11 (s, 3H, C17-H), 1.19 (s, 3H, C16-H),
3
ꢀ
1
.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H), 2.2 (m, 2H, C14-H), 2.23
(dd, 1H, C7-H), 5.53 (d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 6.15 (dd, 1H,
ꢀ
(
s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 2.6 (m, 4H, COCH CH CO),
C3 -H), 6.21 (t, 1H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH),
2
2

S. Arpicco et al. / International Journal of Pharmaceutics 454 (2013) 653–659
655
ꢀ
ꢀ
ꢀ
ꢀ
7
7
.3 (m, 3 -Ph), 7.35 (d, nitrophenyl), 7.4 (m, 3 -NBz), 7.5 (m, 2-OBz),
1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph), 7.4 (m, 3 -NBz), 7.5 (m,
ꢀ
ꢀ
.73 (d, 3 -NBz), 8.1 (d, 2-OBz), 8.26 (d, nitrophenyl).
2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
Compounds 12 and 13, yield 55% 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
ꢀ
2
.2.3. Preparation of 2 -[methoxypoly(ethylene
2
.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.28
glycol)amido-N-metyl-glycine carbamate] paclitaxel derivatives
(
s, PEG OCH ), 3.34 (d, 1H, C3-H), 3.48–4.1 (m, PEG OCH CH O),
ꢀ
3
2
2
Compound (8) was prepared by reaction of 2 -(4-nitrophenyl
4
.17 and 4.28 (d, 2H, C20-H), 4.48 (dd, 1H, C7-H), 4.96 (d, 1H, C5-
carbonate)paclitaxel (5) (15.5 mg, 0.0152 mmol) dissolved in 2 mL
of dry dichloromethane with 8.3 mg (0.0456 mmol) of a solution
of sarcosine tert-butyl-ester in dry dichloromethane containing
triethylamine (85 ␮L, 0.608 ␮mol). The reaction proceeded under
stirring and a nitrogen atmosphere for 2 h at room temperature.
The reaction was washed with 0.1 N HCl, dried with anhydrous
ꢀ
ꢀ
H), 5.65 (d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 6.08 (dd, 1H, C3 -H), 6.21
ꢀ
(t, 1H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph),
ꢀ
ꢀ
7
.4 (m, 3 -NBz), 7.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
Compounds 14 and 15, yield 48% 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.30
s, PEG OCH ), 3.34 (d, 1H, C3-H), 3.42–3.99 (m, PEG OCH CH O),
2
magnesium sulphate and evaporated under reduced pressure.
(
ꢀ
3
2
2
2
-(N-Methyl-glycine carbamate)paclitaxel (9) was obtained by
ꢀ
4
5
1
.17 and 4.28 (d, 2H, C20-H), 4.80 (d, 1H, C2 -H), 4.96 (d, 1H, C5-H),
.42 (dd, 1H, C7-H), 5.67 (d, 1H, C2-H), 5.78 (d, 1H, C3 -H), 6.21 (t,
H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph), 7.4
adding 58.5 ␮L (0.76 ␮mol) of TFA to a dry dichloromethane solu-
tion of 8 (16.2 mg, 0.0152 mmol) and the mixture was allowed to
react for 1 h at room temperature. The mixture was then neutral-
ized with 10% aqueous sodium bicarbonate solution and extracted
with dichloromethane. The organic layer was dried over anhydrous
magnesium sulphate and evaporated under reduced pressure. Yield
9% (10.3 mg) H NMR (CDCl ): ı 1.11 (s, 3H, C17-H), 1.19 (s, 3H,
C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H), 2.2 (m, 2H, C14-H),
.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.2 (s, 3H, 2 -OCONCH ),
.34 (d, 1H, C3-H), 4.17 and 4.28 (d, 2H, C20-H), 4.32 (s, 2H, 2 -
OCONCH ), 4.48 (dd, 1H, C7-H), 4.96 (d, 1H, C5-H), 5.63 (d, 1H,
C2 -H), 5.67 (d, 1H, C2-H), 6.08 (dd, 1H, C3 -H), 6.21 (t, 1H, C13-H),
.27 (s, 1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph), 7.4 (m, 3 -NBz),
ꢀ
ꢀ
ꢀ
ꢀ
(m, 3 -NBz), 7.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
Compounds 16 and 17, yield 50% 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 3.28
s, PEG OCH ), 3.34 (d, 1H, C3-H), 3.45–4.1 (m, PEG OCH CH O),
2
1
6
3
(
3
2
2
4
.17 and 4.28 (d, 2H, C20-H), 4.96 (d, 1H, C5-H), 5.42 (dd, 1H, C7-
ꢀ
2
3
3
ꢀ
ꢀ
ꢀ
H), 5.65 (d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 6.08 (dd, 1H, C3 -H), 6.21
ꢀ
(t, 1H, C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph),
2
ꢀ
ꢀ
7
.4 (m, 3 -NBz), 7.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
ꢀ
ꢀ
ꢀ
ꢀ
6
7
2.3. Water solubility and stability of conjugates
ꢀ
.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
A
solution
of
2-ethoxy-1-(ethoxy-carbonyl)-1,2-
Water solubility was estimated by dissolving appropriate
amounts of conjugates in 0.1 mL of water.
The hydrolysis rate of conjugates was determined at different pH
values, using sodium acetate buffer 0.1 M (pH 5.6), sodium phos-
phate buffer 0.1 M (pH 7.4), sodium borate buffer 0.1 M (pH 9) or
fetal calf serum. Conjugates were dissolved at a concentration of
dihydroquinoline (EEDQ) (3 mg, 0.0121 mmol) in 100 ␮L of
anhydrous dimethylformamide (DMF) was added dropwise to
0 mg (0.0102 mmol) of 9 in 400 ␮L of DMF and the reaction
1
was stirred for 30 min at room temperature. Then, 0.0120 mmol
of m-PEG-NH2 (5 or 20 kDa) in 100 ␮L of anhydrous DMF were
added and the reaction proceeded for 24 h at room temperature.
The course of reaction was followed by HPLC, which showed the
presence of the conjugates 10 and 11. The crude product was
purified by HPLC and the collected fractions were dialyzed against
1
mg/mL. Drug stability was determined by removing portions of
◦
samples from the solutions incubated at 37 C for various periods
of time. 20 ␮L of sample were withdrawn from the solution of the
different buffers and injected into HPLC. To the samples incubated
in serum 200 ␮L of acetonitrile was added to precipitate proteins,
and the resulting solution was vortexed for 30 s and centrifuged
at 330 × g for 5 min. 150 ␮L of supernatant was analyzed by HPLC
using the conditions already described.
water and lyophilized. Yield 63% 1H NMR (CDCl ): ı 1.11 (s, 3H,
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc),
.2 (s, 3H, 2 -OCONCH ), 3.30 (s, PEG OCH ), 3.34 (d, 1H, C3-H),
3
2
3
3
(
(
ꢀ
3
3
.46–3.95 (m, PEG OCH CH O), 4.17 and 4.28 (d, 2H, C20-H), 4.39
2
2
ꢀ
s, 2H, 2 -OCONCH ), 4.48 (dd, 1H, C7-H), 4.96 (d, 1H, C5-H), 5.63
2
2
.4. Tumor cell lines and cell culture
ꢀ
ꢀ
d, 1H, C2 -H), 5.67 (d, 1H, C2-H), 6.08 (dd, 1H, C3 -H), 6.21 (t, 1H,
C13-H), 6.27 (s, 1H, C10-H), 7.07 (d, 1H, NH), 7.3 (m, 3 -Ph), 7.4 (m,
-NBz), 7.5 (m, 2-OBz), 7.73 (d, 3 -NBz), 8.1 (d, 2-OBz).
ꢀ
The cell lines used were MCF-7, a human breast cancer, and
ꢀ
ꢀ
3
HT-29, a human colorectal adenocarcinoma. Both cell lines were
maintained in RPMI 1640 medium containing 10% fetal calf serum
and 1% antibiotics (containing penicillin and streptomycin) in a 5%
2
.2.4. General procedure for the preparation of the PEGylated
compounds
The previously prepared paclitaxel derivatives (2, 5, 6 and 7)
◦
CO2 humidified atmosphere at 37 C.
dissolved in dry DMF were separately reacted with a dry DMF solu-
tion of m-PEG-NH2 (5 and 20 kDa); the PTX:PEG molar ratio was
2.5. Cytotoxicity test
1
:2 for compound 7 and 1:1 for the other derivatives. The reaction
MCF-7 and HT-29 cells, maintained as described above, were
4
mixtures were stirred for 12 h at room temperature. The course of
reactions was followed by HPLC, which showed the presence of the
different conjugates. The crude products were purified by HPLC and
the collected fractions were dialyzed against water and lyophilized.
seeded at 3 × 10 cells/well in microtiter plates and incubated
overnight to allow cellular adhesion. Various dilutions of PTX (in
dimethylsulfoxide) and conjugates (in water) (expressed as pacli-
taxel concentration) were added in triplicate, and incubated for
72 h. The supernatants were removed and the cells washed and
incubated for 16 h with fresh medium containing 1 mCi of l-[4,5-
Compounds 3 and 4, yield 68% 1H NMR (CDCl ): ı 1.11 (s, 3H,
3
C17-H), 1.19 (s, 3H, C16-H), 1.62 (s, 3H, C19-H), 1.76 (s, 3H, C18-H),
2
3
.2 (m, 2H, C14-H), 2.23 (s, 3H, C10-OAc), 2.43 (s, 3H, C4-OAc), 2.63
H]-leucine (58 Ci/mmol). The cells were harvested using with a
ꢀ
(m, 2H, 2 -OCOCH ), 2.95 (m, 2H, CH CON), 3.30 (s, PEG OCH ), 3.34
Skatron Harvester and the incorporated radioactivity was mea-
sured using a Packard-2500 TR Liquid Scintillation Analyzer. The
2
2
3
(
d, 1H, C3-H), 3.45–4.1 (m, PEG OCH CH O), 4.17 and 4.28 (d, 2H,
2
2
ꢀ
3
C20-H), 4.48 (dd, 1H, C7-H), 4.96 (d, 1H, C5-H), 5.51 (d, 1H, C2 -H),
5
results were expressed as percentages of l-[4,5- H]-leucine incor-
ꢀ
.67 (d, 1H, C2-H), 5.80 (d, 1H, C3 -H), 6.21 (t, 1H, C13-H), 6.27 (s,
poration compared to control cultures, background values being

6
56
S. Arpicco et al. / International Journal of Pharmaceutics 454 (2013) 653–659
Fig. 1. Chemical structures of PEG–PTX prodrugs 3, 4 and 10–17.
subtracted. Data are means of three separated experiments, in
which each individual value is the average of triplicate samples
to facilitate the further PEGylation reaction, the carboxyl function
of 2 -succinyl-paclitaxel was activated as N-hydroxysuccinimidil
ꢀ
(
<7% standard error).
derivative (PTX-NHS, 2) using NHS and DCC. PTX-NHS was finally
reacted with m-PEG-NH (5 and 20 kDa) in DMF to give the desired
2
prodrugs (Scheme 1).
3
. Results and discussion
The different PEG-carbamate prodrugs (12–17) were obtained
ꢀ
by preliminary activation of PTX C2 and/or C7 hydroxyl groups
3.1. Chemistry
with 4-nitrophenyl chloroformate (Scheme 2, compounds 5–7) and
consequent reactions with m-PEG-NH .
The schematic structures of the new PEG–PTX prodrugs are
reported in Fig. 1.
2
ꢀ
The activated 2 -carbonate (5) was obtained reacting PTX with
◦
4
-nitrophenyl chloroformate at −35 C, as reported by de Groot
Prodrugs 3 and 4 were prepared starting by the reaction of
ꢀ
(de Groot et al., 2000). The same reaction was performed at room
temperature to prepare the 2 ,7-disubstituted compound (7). We
the 2 -hydroxy function of PTX with succinic anhydride accord-
ꢀ
ing to the procedure of Deutsch (Deutsch et al., 1989). In order

S. Arpicco et al. / International Journal of Pharmaceutics 454 (2013) 653–659
657
Scheme 1. Synthesis of compounds 3 and 4.
were able to obtain the 7-carbonate derivative (6) in a single step
during purification of the 2 ,7-disubstituted carbonate by flash
crude product was left for about 12 h in the column before purifi-
cation. This is an easy and less time-consuming procedure for the
activation of the hydroxyl group of PTX in 7, since most of the
methods reported in literature required the previous protection of
2 -position of PTX (Altstadt et al., 2001; Lee et al., 2005; Ryu et al.,
2008; Wang et al., 2006). Other works describe the preliminary
ꢀ
chromatography; in fact, during the purification we observed the
formation of the 7-(4-nitrophenyl carbonate)paclitaxel obtained
ꢀ
ꢀ
by the hydrolysis of the more reactive compound at 2 -position
ꢀ
from 2 ,7-(4-nitrophenyl biscarbonate)paclitaxel. To this aim the
Scheme 2. Preparation of 4-nitrophenyl carbonate paclitaxel derivatives.

6
58
S. Arpicco et al. / International Journal of Pharmaceutics 454 (2013) 653–659
Scheme 3. Preparation of compounds 10 and 11.
ꢀ
preparation of the 2 ,7-disubstituted derivative and the further
the conjugates did not depend on the PEG’s molecular weight. The
prodrugs 14–17 were highly stable and the PTX release after 72 h
of incubation was about 3–6% at pH 7.4 and 10% (14 and 15) or 3%
(16 and 17) in serum, indicating that we were able to obtain soluble
prodrugs that also provide a slow release of PTX. On the other hand,
compounds 10 and 11 released 100% of PTX after 48 h, but no bac-
catin III formation was observed indicating that the introduction
of a methyl group as steric hindrance provided a protection of the
conjugates. The unhindered conjugates 12 and 13, on the contrary,
gave a rapid release of inactive baccatin III at pH 7.4, that reached
100% after 48 h, so these conjugates were not further evaluated for
their cytotoxicity.
ꢀ
removal of the more chemically labile substituent at the 2 -position,
but cleavage, reaction work up and purification were necessary to
obtain the desired compound (Mathew et al., 1992; Niethammer
et al., 2001; Takahashi et al., 1998). Our approach is more flexible
and permits to obtain the di- or mono-substituted derivative easily
during column purification process.
The preparation of PEG–PTX prodrugs 10 and 11 is reported in
Scheme 3. The introduction of a methyl group as steric hindrance on
the carbamate nitrogen was obtained by reacting 2 -(4-nitrophenyl
carbonate)paclitaxel (5) with sarcosine tert-butyl-ester, then the
protective group was cleaved with TFA and the corresponding
derivative 9 was reacted with m-PEG-NH2 (5 and 20 kDa) in DMF
in presence of EEDQ to give the corresponding PEGylated com-
pounds. We decided to insert a steric hindrance in the 2 -carbamate
PEG–PTX prodrugs because the unhindered derivatives 12 and 13
ꢀ
These experiments clearly demonstrate the higher stability of
carbamate linkage over the ester bond (compounds 3 and 4) and
also indicate that the modification of the more hindered C7 posi-
tion lead to reduction of hydrolysis rate of the conjugates as also
reported by other authors (Greenwald et al., 1995; Sugahara et al.,
2002). The stability was further improved with the introduction of
ꢀ
◦
were highly unstable in buffer solution at pH 7.4 at 37 C. In fact,
in this condition we observed the rapid formation of the inactive
compound baccatin III, due to an addition-elimination sequence
as yet reported by de Groot (de Groot et al., 2000). The authors
demonstrated that the insertion of a methyl group prevented this
unwanted reaction giving more stable derivatives. A quite simi-
lar approach was described by Greenwald in the preparation of
hindered PEG-camptothecin diesters (Greenwald et al., 1998).
ꢀ
PEG at both C2 and C7 positions.
3.3. Cytotoxicity
The cytotoxic activity of PEG–PTX conjugates was evaluated on
MCF-7 and HT-29 human cancer cell lines after 72 h incubation at
◦
Compounds 14–17 were prepared by reacting m-PEG-NH with
37 C. Free PTX was also tested as control. The results, reported in
2
the different carbonate PTX derivatives in order to obtain deriva-
tives characterized by a carbamate linkage and by the presence of
PEG in the C2 and/or C7 positions of the drug.
Table 2, show that PTX was very active and that the prodrugs 3
and 4 display a quite similar toxicity; these data are in accordance
with the stability experiments that indicated that PTX was rapidly
released from the conjugates 3 and 4. On the contrary, an important
decrease in the cytotoxicity (from two to four orders) was observed
in the PEG-carbamate prodrugs, in particular for the compounds
obtained reacting the C7 position of PTX (14 and 15); a reduction
in cytotoxic activity of C7 ester or carbamate derivatives was yet
reported by other research groups (Greenwald et al., 1995, 1996).
Taken together these results show that these PTX derivatives are
very stable and less toxic and that they do not need toxic excipients
ꢀ
All the purified PEG–PTX conjugates were lyophilized and were
◦
stable for several months when stored at −20 C under a nitrogen
atmosphere.
3.2. Water solubility and in vitro stability of PEG–PTX prodrugs
The conjugation of PEG to PTX dramatically increased its aque-
ous solubility; in fact the prodrugs obtained using PEG of 5 kDa
of molecular weight showed a solubility of about 340 mg/mL, in
the case of monosubstituted compounds (3, 10, 12 and 14), and of
Table 1
◦
Hydrolysis rates of PEG–PTX prodrugs in different media at 37 C.
4
60 mg/mL for the disubstituted one (16). The 20 kDa PEG–PTX con-
jugates showed a decrease in solubility values (150 and 180 mg/mL
for mono- (4, 11, 13 and 15) and di-substituted (17), respectively). A
similar behavior was also observed for a reported series of PEG–PTX
prodrugs, in which the water solubility decreased with the increas-
ing of the polymer’s molecular weight (Greenwald et al., 1996).
The in vitro stability of the different PEG–PTX conjugates was
Compound
t½ (h)a
pH 5.6
pH 7.4
pH 9
Serum
3
1
1
1
, 4
5.5
32
ND
ND
3
24
>96(94%)
>96(97%)
1
4
0.5
2.5
0, 11
4, 15
6, 17
b
b
c
b
>96(90%)
>96(90%)
>96(95%)c
>96(97%)b
studied evaluating by HPLC the release of PTX, after incubation at
ND: not determined.
◦
3
7 C in buffer at various pH values (5.6, 7.4 and 9) or in serum;
a
All experiments were done in triplicate. SD: ± 10%.
the results are shown in Table 1. As previously reported by Green-
wald (Greenwald et al., 1996), we also observed that the half-life of
b Percentage of remaining compound after 72 h.
c
Percentage of remaining compound after 48 h.

S. Arpicco et al. / International Journal of Pharmaceutics 454 (2013) 653–659
659
Table 2
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A series of new PEG–PTX prodrugs characterized by different
1
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consuming synthetic approaches. The in vitro studies showed that
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release and low toxicity.
To evaluate the potential therapeutic activity of these new
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Acknowledgement
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