1
60
S. Pedotti et al. / Carbohydrate Polymers 131 (2015) 159–167
a suitable amount of the drug to the target site (Qureshi, Jiang,
Midha, & Skelly, 1998). In many instances intravenous adminis-
tration in the form of a strong alkaline (pHs 10–11) solution of
sodium salt is required in order to obtain a satisfactory response,
but this can cause thrombophlebitis or perivascular inflammation
chromatography (TLC) using aluminium sheets precoated with sil-
ica gel 60 RP-18 F254 (Merck) and detection by UV light and/or
with ethanolic 10% sulfuric acid. Solvents were dried by distilla-
tion according to standard procedure (Perrin, Armarego, & Perrin,
1988) and stored over 4 A˚ molecular sieves activated for at least
◦
(Shojaei, Berner, & Li, 1998.) Moreover, Acy is characterized by a
24 h at 400 C. Double-distilled water was used throughout the
short half-life (2.5–3.3 h) (Susantakumar, Gaur, & Sharma, 2011),
so all therapeutic treatment requires repeated daily administra-
tions (5–6 times), reducing patient compliance. To overcome these
drawbacks, sustained release formulations have been developed,
examples being polymeric nanoparticles (Fresta et al., 2001; Jwala
et al., 2011) or ethosome formulations (Godin & Touitou, 2003;
Zhou, Wei, Zhang, & Wu, 2010). Polymeric or macromolecular pro-
drugs can also be effectively used to increase the bioavailability of
Acy and to obtain the desired sustained release (Giammona, Puglisi,
Cavallaro, Spadaro, & Pitarresi, 1995; Hiramath et al., 2011; Sawdon
study.
A
2
.2. Synthesis of 6 -O-{9-[[2-[(3-
carboxypropionyl)oxy]ethoxy]methyl]guanine}-ˇ-cyclodextrin
conjugate (ˇ-CyDAcySucc, 3)
Sodium
methoxy]ethoxy}-4-oxobutanoate
.88 mmol) (Colla, De Clercq, Busson, & Vanderhaeghe, 1983) was
added to a solution of 6A-O-(p-toluenesulfonyl)--cyclodextrin
-CyDOTs, 2) (1.13 g, 0.88 mmol) (Djedaini-Pilard, Désalos &
4-{2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)
(AcySuccNa, 1) (0.30 g,
0
&
Peng, 2014). The conjugation of Acy with natural -CyD could be
(
an effective approach for producing a sustained release of the drug
making it suitable for oral or other routes of administration such as
intra-pulmonary or intra-vitreal, etc. Acy is widely used in the treat-
ment of various ocular diseases such as herpes simplex keratitis
and acute retinal necrosis; in the latter case intra-vitreal admin-
istration of the drug could be more efficacious than intravenous
administration in the attempt to reach therapeutical concentra-
tions in the intraocular region (Damico et al., 2012). However, due
to the great invasiveness of the intra-vitreal injections, frequent
administrations cannot be considered. A prolonged release of the
drug could be of noteworthy therapeutical importance in this case.
This work is a preliminary studies to synthetize Acy--CyD conju-
gate and evaluate its hydrolysis in media simulating physiological
environments, in order to explore the potentiality of this prodrug
for use through various routes of administration (oral, intraocular,
inhalatory and so on). During experimentation Acy was selectively
linked to one primary hydroxyl of -CyD by ester linkage using
succinic moiety as a spacer and was characterized through NMR
studies. Two-dimensional NMR experiments were used to inves-
tigate the spatial arrangement of the conjugate. Water solubility,
dissolution rate and the octanol/water partition coefficient of the
conjugate were evaluated by comparison with free Acy. Chemical
and enzymatic hydrolysis studies were performed to investigate
the potentiality of the conjugates as sustained release systems for
Acy. For enzymatic hydrolysis we used the isoenzymes of porcine
liver esterase (PLE), a non-specific carboxylase in wide use for the
last 30 years and highly active both as an esterase and amidase (Ge
et al., 2013; Huang et al., 1996). A large number of papers report the
use of the isoenzymes of PLE to study the hydrolysis of the prodrug,
co-drug or macromolecular conjugate developed for various routes
of administration, including parenteral (Lau, Heard, & White, 2013;
Lee et al., 2011; Swartz, Zhang, Valeriote, Chen, & Shaw, 2013; Wang
et al., 2012; Wu, Shaw, Dubaisi, Valeriote, & Li, 2014).
Perly, 1993) in DMF (20 mL), and the resulting mixture was stirred
at 100 C for 24 h. The solvent was then removed under reduced
◦
pressure, yielding a residue which was washed with water (50 mL)
to remove the unreacted -CyDOTs 2. After filtration, the obtained
filtrate containing -CyDAcySucc 3 was concentrated under vac-
uum and the resulting crude product was purified by reverse phase
chromatography on Lichroprep RP-18 (gradient eluent: from 0% to
3
5% of methanol in water). The fractions containing -CyDAcySucc
3
were collected and the eluent was removed under reduced
pressure. Finally, the -CyDAcySucc 3 obtained was lyophilized to
yield 0.57 g (0.39 mmol, yield 45%).
◦
-CyDAcySucc 3; pale yellow crystals; m.p. 249–250 C; Rf, 0.27
(
propanol/H O/NH OH/AcOEt, 5:3:2:1, v/v/v/v); ˛ = +133 (c = 1,
2 4
1
H O); H NMR (500 MHz, D O): 2.64 (m, 2H, COCH ), 2.72 (m, 2H,
2
2
2
COCH ), 3.54 (t, 1H, -CyD H-4A), 3.65–3.73 (m, 13H, -CyD H-2
2
and H-4), 3.76 (m, 2H, OCH CH ), 3.83–3.98 (m, 25H, -CyD H-6, H-
2
2
5
and H-3), 4.10 (m, 1H, -CyD H-6A), 4.18 (m, 2H, OCH CH ), 4.79
2 2
(
d, 1H, -CyD H-6A), 5.09–5.10 (m, 7H, -CyD H-1), 5.56 (s, 2H,
13
N-CH ), 7.89 (s, 1H, H-8); C NMR (125 MHz, D O): ␦ 29.0, 29.5
2
2
(
COCH ), 60.1 (C-6), 64.0 (OCH CH ), 64.6 (OCH CH ), 66.7 (C-6A),
2 2 2 2 2
7
0.7 (C-5), 72.0 (C-3), 72.7 (N-CH ), 73.3 (C-2), 81.7 (C-4), 102.1
2
ꢀ
ꢀ
ꢀ
ꢀ
(
(
(
C-1), 116.1 (C-3 ), 140.0 (C-5 ), 152.3 (C-4 ), 154.2 (C-2 ), 159.0
C-6 ), 172.0 (C O), 174.1 (C O); FAB MS (glycerol/water), 1443
M+1) m/z.
ꢀ
2.3. Apparatus
Fast atom bombardment (FAB) mass spectra were recorded
on
a VG ZAB-25E spectrometer, in a positive mode using
glycerol water. The chromatographic analyses were performed
using a HPLC system Varian ProStar model 230 (Varian, Milan,
Italy) using a reverse-phase column (C18) Waters Symmetry
(5 m × 4.6 mm × 15 cm). The HPLC apparatus was equipped with
an auto-sampler Varian model 410 and Galaxie software for data
elaboration. The mobile phase consisted of a mixture 88/12 (v/v) of
acetonitrile/ammonium acetate buffer 20 mM (pH 3.7). All analyses
were carried out at room temperature, at a flow rate of 1.0 mL/min.
Twenty microlitres of each sample were injected and the column
effluent was monitored continuously at 254 nm. The amount of Acy
was calculated by reporting the peak area of a sample on a standard
calibration curve in the range between 0.4 and 10.0 g/mL of Acy
2
. Materials and methods
2.1. Materials

-Cyclodextrin (-CyD) was purchased from Cyclolab R & D
Laboratory (Budapest, Hungary) and used after desiccation in a vac-
uum with phosphoric anhydride for 24 h at 90 C. Acyclovir (Acy),
anhydrous N,N-dimethylformamide (DMF), anhydrous pyridine
◦
2
1
(r = 0.9922). H NMR spectra were recorded with a Varian VnmrJ
instrument at 500 MHz in the aforementioned solvent. All sam-
(
Py), succinic anhydride, triethylamine, thionyl chloride, porcine
ples were solubilized in D O; no internal standard was added to
liver esterase (PLE) (EC 3.1.1.1., lyophilized powder, ≥15 units/mg
2
the samples in order to avoid its interaction with the -CyD cav-
solid) and phosphate buffer solutions (PBS) were Sigma–Aldrich
ity. The residual sign of HOD at 4.83 ppm was used as reference
(
Milano, Italy) products. Lichroprep RP-18 (E. Merck, 40–63 m)
(
Ivanov, Salvatierra, & Jaime, 1996). 13C NMR spectra were recorded
was used for reverse-phase flash chromatography with differ-
ent eluent mixtures. All reactions were followed by thin-layer
at 125 MHz. Chemical shifts are given in ppm (␦).