Synthesis of pro-prodrugs l-lysine based for 5-aminosalicylic acid and 6-mercaptopurine colon specific release

Article abstract of DOI:10.1016/j.ijpharm.2011.09.001

The aim of this work is to design, prepare and characterize l-lysine based prodrugs capable of targeting 6-mercaptopurine to the colon, an anti-tumor and immunosuppressant drug, and 5-aminosalicylic acid (5-ASA), drug of choice for inflammatory bowel disease (IBD). More specifically, Nε-feruloyl-S-(6- purinyl)-l-lysine and Nε-acryloyl-S-(6-purinyl)-l-lysine were synthesized and then characterized by FT-IR, ¹H-NMR and GC/MS spectroscopies. The ability of feruloyl derivative in inhibiting lipid peroxidation in rat liver microsomal membranes, induced in vitro by tert-butyl hydroperoxide as source of free radicals, was evaluated. Moreover, Nε-acryloyl-S-(6-purinyl)-l-lysine, polymerizable prodrug, was used to microspheres realization for 5-ASA release. These lasts, obtained by emulsion inverse technique, were characterized by light scattering and scanning electron microscopy (SEM) analysis. The microspheres equilibrium swelling degree was evaluated and showed good swelling behaviour in simulating colonic fluids. Results confirm the possibility that the application range of l-lysine prodrug can be extended to the treatment of intestinal diseases whose conventional therapy envisages medications with serious side effects that, thanks to this new strategy, can be minimized in an optimal way.

Full text of DOI:10.1016/j.ijpharm.2011.09.001

International Journal of Pharmaceutics 420 (2011) 290–296
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Synthesis of pro-prodrugs l-lysine based for 5-aminosalicylic acid and
6-mercaptopurine colon specific release
Sonia Trombino, Roberta Cassano∗, Alessia Cilea, Teresa Ferrarelli, Rita Muzzalupo, Nevio Picci
Department of Pharmaceutical Sciences, University of Calabria, 87036 Arcavacata di Rende, Cosenza, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this work is to design, prepare and characterize l-lysine based prodrugs capable of target-
ing 6-mercaptopurine to the colon, an anti-tumor and immunosuppressant drug, and 5-aminosalicylic
acid (5-ASA), drug of choice for inflammatory bowel disease (IBD). More specifically, N␧-feruloyl-S-(6-
purinyl)-l-lysine and N␧-acryloyl-S-(6-purinyl)-l-lysine were synthesized and then characterized by
FT-IR, 1H-NMR and GC/MS spectroscopies. The ability of feruloyl derivative in inhibiting lipid peroxi-
dation in rat liver microsomal membranes, induced in vitro by tert-butyl hydroperoxide as source of free
radicals, was evaluated. Moreover, N␧-acryloyl-S-(6-purinyl)-l-lysine, polymerizable prodrug, was used
to microspheres realization for 5-ASA release. These lasts, obtained by emulsion inverse technique, were
characterized by light scattering and scanning electron microscopy (SEM) analysis. The microspheres
equilibrium swelling degree was evaluated and showed good swelling behaviour in simulating colonic
fluids. Results confirm the possibility that the application range of l-lysine prodrug can be extended to
the treatment of intestinal diseases whose conventional therapy envisages medications with serious side
effects that, thanks to this new strategy, can be minimized in an optimal way.
Received 17 January 2011
Accepted 1 September 2011
Available online 8 September 2011
Keywords:
Prodrug
trans-Ferulic acid
Methacrylic acid
6-Mercaptopurine
Microspheres
Lipid peroxidation
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Particular interest in recent years has been focused on the activ-
ity of azoreductase that catalyzes the reduction of a diazo bond
During the last few decades, the pharmaceutical research has
focused its attention on the development of formulations that can
release the drug in the body in a controlled and specific manner
to decrease the number of daily dosing, improve patient compli-
ance and minimize adverse reactions. These formulations, defined
as controlled release systems or Drug Delivery System outweigh the
disadvantages associated with conventional treatment systems.
The creation of pharmaceutical forms able of selective colon
release is gaining particular interest, above all, for the treatment of
local diseases such as ulcerative colitis, Crohn’s disease, rectal can-
cer, and irritable bowel syndrome (Friend, 2005; Gazzaniga et al.,
2006; Jain et al., 2007).
A prodrug should be sufficiently hydrophilic and bulky to min-
imize the absorption in the stomach and small intestine, but must
become more lipophilic in the colon to be absorbed. Due to the
wide variety of enzymes produced by intestinal flora at this level,
generally anaerobic bacteria responsible of different enzymes pro-
duction such as esterases, amidase, nitroreductase, azoreductase,
deaminase, urea dehydroxylase, it has been possible to prepare
prodrugs subject to enzymatic cleavage (Kinget et al., 1998).
with regeneration of two primary amino groups and glycosidases
that catalyzes the hydrolysis of glycosidic bonds. The colon-specific
prodrug for excellence, presenting a diazo bond, is sulfasalazine;
however, other prodrugs were developed by coupling, through a
␤-glycosidic bond, an hydrophilizing residue (glucose or galactose)
to steroids such as dexamethasone, prednisolone, hydrocortisone
and fluorocortisone (Van den Mooter, 2006). The sugar presence
makes the therapeutic agent sufficiently polar to suffer minimal
absorption in the small intestine, while in the colon, thanks to
the bacterial glycosidases activity, the polar fraction releases the
steroid, which can carry out its action (Rubinstein, 2005; Makins
and Cowan, 2001; Taxonera et al., 2009). The prodrugs based on
amino acids are very effective in the treatment of IBD (inflamma-
tory bowel disease) for the presence of very chemically reactive
groups that are, at the same time, sites of various enzymes action
(esterase, amidase, azoreductase); in particular, l-lysine possesses
three sites of functionalization, two amino groups and a carboxyl
one. For this reason, a pro-prodrug l-lysine based, binding 5-ASA
and trans-ferulic acid as active substances was recently prepared
in order to optimize biocompatibility and allow the drug colon-
specific release (Cassano et al., 2009). Functionalization occurs at
the level of the amino group in ␧ position and the amino group
directly linked to the chiral carbon with the formation of amide
or diazo bonds. The presence of these multiple sites allowed us to
∗
Corresponding author. Tel.: +39 984493203; fax: +39 984493163.
E-mail address: roberta.cassano@unical.it (R. Cassano).
0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.09.001

S. Trombino et al. / International Journal of Pharmaceutics 420 (2011) 290–296
291
Table 1
Reaction conditions for 3a and 3b synthesis.
Reaction conditions for 3a
Reaction conditions for 3b
Reagent
Amount (g or ml)
mmol
Reagent
Amount (g or ml)
mmol
135.5
MKC
2a
DCC
DMAP
6-MP
8.30
0.085
FC
2b
DCC
DMAP
6-MP
16.51
0.308
0.220
0.012
0.650
0.065
0.070
0.004
0.208
0.305
0.954
3.4 × 10−4
1.051
0.095
3.816
0.030
1.220
design pro-prodrugs for the site-specific release of various pharma-
cologically active molecules. In particular, objective of this study,
was the realization of both pro-prodrugs l-lysine based contain-
ing acrylic moieties and mercaptopurine or trans-ferulic acid and
mercaptopurine. This latter was submitted to antioxidant activity
evaluation. On the other hand, the polymerizable derivative was
employed to prepare microspheres useful as carrier of other colon
specific drugs and their swelling and release behaviour were also
evaluated.
a value of 90 ◦C. Then, 33.3 g (269 mmol) of copper carbon-
ate (CuCO3) were added slowly to obtain intermediate 1a–1b
(Scheme 1). Subsequently, 290 ml (3.945 mmol) of acetone and
a potassium hydroxide aqueous solution (15.37 g, 274 mmol of
KOH in 137 ml of distilled water) were added, under stirring still.
Then, methacryloyl chloride (MKC) or trans-ferulic acid acyl chlo-
ride (FC) prepared as described in our previous work (Cassano
et al., 2009) (Table 1) were added and temperature given to
3 ◦C. Then, a potassium hydroxide aqueous solution (17.26 g,
308 mmol of KOH in 154.8 ml of distilled water) was added for
four times every 5 min and the reaction was left for 12 h. After
that, the obtained precipitate was filtered and washed with dis-
tilled water, methanol and diethyl ether, drying the product after
each washing. Obtained products (1a–1b) were blue colored and
analyzed though FT-IR spectroscopy (Table 2). Yield: 1a 78%; 1b
81%.
2. Materials and methods
2.1. Materials
All solvents of analytical grade, were purchased from
Carlo Erba Reagents (Milan, Italy): acetone, chloroform,
dichloromethane, ethanol, ethyl ether, isopropanol, methanol,
N,N-dimethylformamide (DMF), n-hexane and tetrahydro-
furan (THF). N-hexane, chloroform, N,N-dimethylformamide
(DMF) and tetrahydrofuran were purified by standard proce-
dures. Monohydrochloride l-lysine (MW = 182.65, purity ≥ 98%),
6-mercaptopurine monohydrate (MW = 170.19, purity 98%),
methacryloyl chloride, basic copper carbonate, KOH (pellets),
8-quinolinol, sorbitan trioleate (Span 85), polyoxyethylene sorbi-
tan trioleate (Tween 85) N,N,Nꢀ,Nꢀ-tetramethylethylenediamine
(TMEDA), N,N-dimethylacrylamide (DMAA), ammonium persulfate
(APS), dicyclohexylcarbodiimide (DCC), dimethylaminopyridine
(DMAP) and thionyl chloride (SOCl2) were purchased from
Sigma–Aldrich (Sigma Chemical Co, St. Louis, MO, USA).
Afterwards, 10 g (21 mmol) of products 1a–1b were dissolved
in 138 ml of distilled water, then a solution of chloroform con-
taining the decomplexing agent, 8-quinolinol (3.64 g, 25 mmol of
8-quinolinol dissolved in 138 ml of chloroform), was added and the
reaction left to reflux for more than 12 h under stirring. Extrac-
tion with chloroform furnished an aqueous solution containing
decomplexed products (product 2a–2b) that were purified through
recrystallization in THF and characterized by FT-IR, GC/MS and 1
H
NMR spectroscopies (Table 2). Esterification of product 2 led to the
desired pro-prodrug. Yield: 2a 94%; 2b 88%
The reaction was conducted according to the procedure
reported in the literature (Matthew et al., 2007). Products
2a–2b (Table 1) were dissolved in the minimum amount of
dimethylformamide (DMF) previously purified. After dissolu-
tion, dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine
(DMAP) were added (Table 1) and the reaction mixture was
brought to reflux at 0 ◦C and left for 5 min under stirring. Then, 6-
mercaptopurine (6-MP) was slowly added dropwise (Table 1) after
its dissolution in the minimum amount of DMF. The reaction was
carried out with stochiometric ratio 1:4 for derivatized l-lysine and
6-mercaptopurine, respectively. The system was left under reflux
for 72 h at the temperature of 50–60 ◦C. The reaction was constantly
monitored by TLC. Finally, DMF was removed by distillation and the
crude product was washed with methanol, to remove dicyclohexy-
lurea (DCU), then dried and purified by chromatographic column.
The obtained product (3a–3b) was carefully analyzed by GC/MS and
NMR spectrometry (Table 2). Yield: 3a 65%; 3b 70%.
2.2. Methods
The infrared spectra were obtained from KBr pellets using a FT-
IR spectrometer Perkin-Elmer 1720, in the range 4000–400 cm−1
(number of scans 16). 1H NMR spectra were processed using a
spectrometer Burker VM30; chemical shifts are expressed in ı
and referred to the solvent. The structures of the compounds syn-
thesized were confirmed also by GC–MS Hewlett Packard 5972.
UV–vis spectra were realized through a UV-530 JASCO spectropho-
tometer. The light scattering was performed with a Brookhaven 90
plus particle size analyzer. The samples were lyophilized utilizing
a “freezing–drying” Micro module apparatus, Edwards. Scanning
electron microscopy (SEM) photographs of the microspheres were
obtained with a JEOL JSMT 300 A; the surface of the samples was
made conductive by deposition of a gold layer on the samples in a
vacuum chamber.
2.4. Preparation of microspheres
N␧-acryloyl-S-(6-purinyl)-l-lysine (3a) based
2.3. Synthesis of prodrugs N␧-acryloyl-S-(6-purinyl)-l-lysine
(3a) and N␧-feruloyl-S-(6-purinyl)-l-lysine (3b)
A round-bottomed cylindrical glass reaction vessel fitted with
an anchor-type stirrer of suitable capacity (100–150 ml), was
flamed in a nitrogen flow and after cooling immersed in a
bath thermostatically controlled at 40 ◦C. Dry n-hexane (20 ml)
and chloroform (18 ml) were introduced as dispersant phase,
then kept under mechanical agitation for 30 min. Polymerization
reaction was conducted in according to the procedure reported
in literature (Freiberg and Zhu, 2004). A fixed amount (42 mg,
The reaction was conducted according to the procedure
reported in the literature (Sinha and Kumria, 2001). Briefly,
50 g (274 mmol) of monohydrochloride l-lysine were dissolved
in 600 ml of distilled water and the reaction mixture was left
under reflux and magnetic stirring until the temperature reached

292
S. Trombino et al. / International Journal of Pharmaceutics 420 (2011) 290–296
O
H2N
O
O
x
CaCO3 Cu(OH)2
H2N
2
NH2
OH
H2O, 90°C
NH2
Cu
intermediate 1a-1b
NH2
O
NH2
acetone, KOH water
solution, 3°C
MKC or FC
O
OMe
O
H
N
HO
O
H
N
O
O
O
NH2
O
NH2
or
Cu
Cu
O
NH2
O
NH2
O
N
H
OMe
O
N
H
O
1a
O
OH
1b
OH
N
H2O, CHCl3
OMe
O
H
N
HO
O
H
N
OH
O
NH2
O
H
2b
O
NH2
2a
SH
N
N
DCC, DMAP, DMF
N
H
0°C, 5 min/
50-60°C, 72 h
H2N
N
OMe
N
N
N
O
N
HO
H
N
O
N
H
N
N
S
N
S
N
NH2
O
NH2
NH2
O
NH2
3a
3b
Scheme 1. Synthetic routes for 3a and 3b.
Table 2
FT-IR, GC/MS and 1H NMR data.
Product
M/Z
Wavenumber ꢀ (cm−1
)
Chemical shift (ı) ppm
2a
55 (100%)
1657 (–CONH), 1617 (–COOH), 917 (C CH2)
1735 (–COSR), 1651 (–CONH), 916 (–C CH2)
(D2O) 5.40 (1H, sb), 5.16 (1H, sb), 3.33 (1H, m), 2.76
(2H, t), 1.66 (3H, s), 1.08–1.60 (6H, m)
(D2O) 8.66 (1H, s), 8.46 (1H, s), 5.53 (1H, sb), 5.17 (1H,
sb), 3.45 (1H, sb), 3.01 (2H, m), 2.10 (3H, s), 1.50–1.90
(6H, m)
(CD3OD) 7.75 (1H, sb), 7.27 (2H, m), 6.85 (1H, d), 6.60
(1H, sb), 3.90 (3H, s), 3.36 (1H, m), 2.78 (2H, t),
1.12–1.72 (6H, m)
(D2O) 8.43 (1H, s), 8.25 (1H, s), 7.77 (1H, m), 7.63 (1H,
m), 7.14 (1H, m), 6.88 (1H, m), 6.52 (1H, m), 3.70 (3H,
s), 3.42 (1H, sb), 3.07 (2H, m), 1.50–1.90 (6H, m).
3a
2b
3b
55 (7%), 69 (100%), 367 (2%)
222 (100%), 77 (6%), 177 (71%)
55 (38%), 368 (100%)
1711 (–CONH)
1707 (–COSR), 1656 (–CONH)

S. Trombino et al. / International Journal of Pharmaceutics 420 (2011) 290–296
293
0.114 mmol) of 3 was dissolved in 4 ml of distilled water, sonicated
for a few minutes to obtain a solution, then added with 7.21 ␮l
(0.057 mmol) of the comonomer N,N-dimethylacrylamide (DMAA)
and 800 mg of ammonium persulfate which acts as an initiator
radical.
Here Ci is the concentration of drug in solution before the load-
ing study and Co is the concentration of drug in solution after the
loading study.
2.8. In vitro drug release from microparticles
The density of the organic phase was adjusted by the addi-
tion of the two organic solvent so that the aqueous phase sank
slowly when stirring stopped. After that, it was first added of
sorbitan trioleate (Span 85, 150 ␮l) and polyoxyethylene sorbitan
trioleate (Tween 20, 150 ␮l). After 10 min, 150 ␮l of N,N,Nꢀ,Nꢀ-
tetramethyl diethylamine (TMEDA) were added and reaction
was left under stirring for 3 h. Microspheres so obtained were
filtered, washed with 100 ml portions of 2-propanol, ethanol, ace-
tone and diethyl ether and dried overnight under vacuum. Their
characterization was effected by optical microscopy and FT-IR spec-
troscopy.
Dried microspheres (10 mg) were dispersed in 6 ml of swelling
media (acidic solution pH 1.2, simulated gastric fluid; phos-
phate buffer pH 7.8, simulated small intestinal fluid; phosphate
buffer pH 6.5, simulated colonic fluid). The test tubes were
maintained at 37 ◦C in an horizontal-shaking bath and shaked
at a rate of 100 rpm. At predetermined intervals, the samples
were centrifuged, 5 ml of supernatant was removed and the
medium was replaced with fresh solutions to maintain the same
total volume throughout the study. The concentration of 5-
aminosalicylic acid was determined by UV spectrophotometry at
fixed wavelengths depending on the different swelling media. In
particular, the release profile in the acidic solution at pH = 1.2
was recorded at 204 nm (ε = 4566 l mol−1 cm−1); the release pro-
file in the phosphate buffer at pH 6.5 was recorded at 198 nm
(ε = 2840 l mol−1 cm−1); the release profile in the phosphate buffer
at pH 7.8 was recorded at 196 nm (ε = 19 000 l mol−1 cm−1). Each
in vitro release study was repeated in triplicate. Drug release was
calculated in terms of cumulative release (% drug release) (Khan
et al., 1999).
2.5. Size distribution analysis
The size of microparticles was determined by dynamic light
scattering (DLLS) using a 90 Plus Particle Size Analyzer (Brookhaven
Instruments Corporation, New York, USA) at 25 ◦C by measuring the
autocorrelation function at 90◦ scattering angle. Cells were filled
with 100 ml of sample solution and diluted to 4 ml with filtered
(0.22 mm) water. The polydispersity index (PI) indicating the mea-
sure of the distribution of nanoparticle population (Koppel, 1972)
was also determined. Six separate measurements were made to
derive the average. Data were fitted by the method of inverse
“Laplace transformation” and Contin (Provencher, 1982a,b).
2.9. Evaluation of N␧-feruloyl-S-(6-purinyl)-l-lysine (3b)
antioxidant activity
In order to evaluate its antioxidant activity, product 6 was
subjected to the malondialdehyde (MDA) test in rat liver micro-
somal membranes (Trombino et al., 2008). Liver microsomes were
prepared from Wistar rats by tissue homogenisation with 5 vol-
umes of ice-cold, 0.25 M sucrose, containing 5 mM HEPES, 0.5 mM
EDTA, at pH 7.5, in a Potter–Elvehjem homogenizer (Tatum et al.,
1990). Microsomal membranes were isolated by removal of the
nuclear fraction at 8000 × g for 10 min and removal of the mito-
chondrial fraction at 18 000 × g for 10 min. The microsomal fraction
was sedimented at 105 000 × g for 60 min, and the fraction was
washed once in 0.15 M KCl and collected again at 105 000 × g for
30 min (Ohta et al., 1997). The membranes, suspended in 0.1 M
potassium phosphate buffer, at pH 7.5, were stored at −80 ◦C.
Microsomal proteins were determined by the Bio-Rad method
(Bradford, 1976). The microsomes were suspended in 0.1 M phos-
phate buffer at pH 7.5. The complex 6 was quickly added to
microsomes. Microsomes were added to the amount of tert-BOOH
to reach a final concentration of 0.25 mM. Microsomal suspensions,
gently suspended using a Dounce homogeniser, were incubated
at 37 ◦C in a shaking bath under air and in the dark for 1 h.
Thereafter, aliquots of 1 mL of microsomal suspension (0.5 mg of
proteins) were mixed with 3 mL of 0.5% TCA and 0.5 mL of TBA
solution (two parts 0.4% TBA in 0.2 M HCl and one part distilled
water) and 0.07 mL of 0.2% BHT in 95% ethanol. Samples were
then incubated in a 90 ◦C bath for 45 min. After incubation, the
TBA–MDA complex was extracted with 3 mL of isobutyl alcohol.
The absorbance of the extracts was measured by the use of UV
spectrophotometry at 535 nm, and the results were expressed
as nmol per mg of protein, using an extinction coefficient of
2.6. Swelling studies
The swelling behaviour was investigated in order to check the
hydrophilic affinity of spherical microparticles. Typically, aliquots
(50 mg) of dried materials were placed in a tared 5-ml sintered
glass filter (Ø 10 mm; porosity G3), weighed, and left to swell by
immersing the filter in a beaker containing the swelling media
(acidic solution pH 1.2, simulated gastric fluid; phosphate buffer
pH 6.5, simulated colonic fluid; phosphate buffer pH 7.8, simulated
small intestinal fluid) (Khan et al., 1999). At predetermined times
(1, 6, 12 and 24 h), the excess of water was removed by percola-
tion and then the filter was centrifuged at 3500 rpm for 15 min
and weighed. The filter tare was determined after centrifugation
with only water. The weights recorded at different times were aver-
aged and used to give the equilibrium swelling degree (Wt(%)) by
Eq. (1) where Ws and Wd are the weights of swollen and dried
microspheres, respectively. Each experiment was carried out in
triplicate and the results were in agreement within 4% standard
error.
Ws − Wd
Wt (%) =
× 100
(1)
Ws
2.7. Incorporation of drug into preformed microspheres
Incorporation of drugs into microspheres was performed by
soaking procedure as follows: 150 mg of preformed empty micro-
spheres (prepared as described above) were wetted with 20 ml of
water in a concentrated drug solution (0.002 mg/ml). After 3 days,
under slow stirring at 37 ◦C, the microspheres were filtered and
dried at reduced pressure in presence of P2O5 to constant weight
(Pitarresi et al., 2004). The loading efficiency percent (LE, %) of all
samples were determined by spectrophotometric analysis of fil-
tered solvent in according to Eq. (2):
1.56 × 10−5 l m−1 cm−1
.
3. Results and discussion
Literature data concerning studies of carrier molecules useful
in site-specific prodrugs preparation, show an increased interest
toward these materials, because they combine the biocompat-
ibility with the site-specificity against target organs. For these
Ci − Co
LE (%) =
× 100
(2)
Ci

294
S. Trombino et al. / International Journal of Pharmaceutics 420 (2011) 290–296
Fig. 1. SEM micrographs.
reasons, in the present work, we synthesized and characterized
prodrugs l-lysine based. Due to the presence of three function-
alizable groups (one carboxylic- and two amino-groups) l-lysine
was derivatized both with 6-mercaptopurine, immunosuppressive
and anticancer drug, and trans-ferulic or methacrylic acid. In addi-
tion, the methacrylic prodrug was polymerized with a specific
comonomer to obtain microspheres able to incorporate other colon
specific active substances. The microspheres swelling behaviour at
different pH was evaluated with great results above all at sim-
ulating colonic fluids. Moreover the release profile of prepared
microspheres was studied according to some studies concerning
the variation of the pH value throughout the GI tract; in fact
important investigations using sensitive and reliable equipments
contradict the traditional view and provide evidence of a fall in pH
at the GI region between ileum and colon. Apparently, the colon has
a lower pH value (6.5) than the small intestine (7.0–7.8), and the
jejunal region of some individuals has a higher pH (range 6.1–7.2)
than the small intestine or colon of other individuals (Evans et al.,
1988). On the other hand, the ferulate derivative was submitted to
antioxidant activity evaluation.
Fig. 1a the spherical shape of our microspheres are evident. Fig. 1b
shows a spongy surface characterized by a high degree of porosity.
3.3. Microspheres swelling behaviour
Investigation of the applicability of these microparticles in con-
trolled release was done by studying their swelling behaviour.
The value of contained water percentage was determined in aque-
ous media which simulates some biological fluids, such as gastric
(pH 1.2), intestinal (pH 7.8) and colonic (pH 6.5) at 37 ◦C. The
data reported in Table 3 illustrate the water uptake, in grams per
grams of dry copolymer, for each composition and pH studied. The
prepared materials show different water affinity at the different
pH values. In particular, at pH 1.2 there is a considerable lower-
ing of the water affinity due to acidic groups unionized at this
pH value. When the pH is 6.5, the water content is greater than
that found at pH 1.2. It is possible to explain this behaviour as a
consequence of electrostatic repulsions between polymeric chains
due to the increase of dissociated groups at pH 6.5. Microspheres
showed a best swelling behaviour in solutions simulating colonic
environment. With the time, this effects increase in an extraordi-
nary manner, rather than to decrease with the maximum swelling
effect at 24 h. These results are very encouraging and confirm the
application of the prodrug in the colonic tract for the treatment of
IBD.
3.1. Microspheres preparation and characterization
Chemical groups susceptible of radical polymerization were
introduced onto l-lysine by acylation with MA. Under reac-
tion condition only l-lysine ␧-amino groups react with acylating
agent. Microspheres were synthesized by copolymerization of
N␧-acryloyl-S-(6-purinyl)-l-lysine (3a) with a comonomer such
as DMAA. The reaction was started using TMEDA and ammo-
nium persulfate as initiator system. The obtained materials were
characterized by Fourier Transform IR spectrometry, particle size
distribution analysis and morphological analysis. The FT-IR spec-
tra of all samples shows the disappearance of bands at 944
and 934 cm−1 awardable to C–C double bond of methacrylic
groups.
3.4. Evaluation of the in vitro drug release from microparticles
In order to estimate the ability of prepared matrices to release
encapsulated drug, the beads were loaded with 5-aminosalicylic
acid by soaking procedure and the loading efficiency (LE, %) was
determined by spectrophotometric analysis such as reported in
experimental part. The experimental data, reported in Fig. 2,
are interesting. The 5-aminosalicylic acid was almost completely
loaded on the polymeric beads (LE (%) = 78.6). It is possible to
explain this behaviour as consequence of strong interactions
between polymeric matrix and acidic drugs, such as 5-ASA. Drug
3.2. Particle sizes and scanning electron microscopy (SEM)
The light scattering analysis confirmed microparticle presence
with a good polydispersity index (0.119) and an average diameter
of 13 ␮m approximately.
Morphological analysis of the microspheres was carried out
using SEM, which is the most widely used of the surface analytical
techniques. SEM represents an invaluable tool for studying sur-
face topography and failure analysis. The technique, which enables
qualitative three dimensional (3-D) imaging of surface properties,
clearly illustrated that the microparticles had a spherical shape. In
Table 3
Microsphere swelling behaviour.
Time
Swelling (˛ %)
pH 1.2
pH 6.5
pH 7.8
1 h
6 h
12 h
24 h
270.2%
254.8%
273%
342.5%
691.9%
803%
284.3%
302%
616.2%
841%
283%
1067%

S. Trombino et al. / International Journal of Pharmaceutics 420 (2011) 290–296
295
characteristic that allow to have a high potential in biomedical field
minimizing adverse reaction of conventional therapies.
The effects of both pro-prodrug and l-lysine ferulate on the lipid
peroxidation were time-dependent with the preservation of their
antioxidant activity up to 2 h (Fig. 3).
4. Conclusions
Prodrugs l-lysine based containing purinic moieties were suc-
cessfully obtained and characterized by common spectroscopy
techniques as FT-IR, GC/MS and 1H NMR. Using polymerizable or
antioxidant substances two prodrugs were prepared. The polymer-
izable one was used to prepare microspheres useful as carrier of
other colon specific drugs like 5-ASA. Scanning electron microscopy
showed a spherical shape and a spongy surface. The light scatter-
ing technique allowed to effect dimensional analysis. Microspheres
swelling studies were also conducted and showed a very good
swelling behaviour in simulating colonic fluids. On the other hand,
the ability of feruloyl derivative in inhibiting lipid peroxidation
in rat liver microsomal membranes induced in vitro by tert-butyl
hydroperoxide as source of free radicals was evaluated. Results
revealed an important capacity to overthrow the intrinsic pro-
oxidant effect of 6-MP. Finally, our prodrugs could have high
potential in biomedical field, in particular in the tumors treat-
ment, targeting 6-mercaptopurine to the colon and outweighing
the disadvantages associated with conventional treatment sys-
tems.
Fig. 2. 5-ASA release evaluation. (For interpretation of the references to color in this
artwork, the reader is referred to the web version of this article.)
release profile was determined by spectrophotometric analysis.
The drug release was expressed as the percent of drug delivered,
related to the effectively entrapped total dose, as a function of time.
We have carried out the in vitro release studies at 37 ◦C. The exper-
imental data showed an increase of 5-ASA release at pH 6.5; the
drug is released in very low amounts both in the stomach that in
the small intestine.
Acknowledgement
3.5. Evaluation of N␧-feruloyl-S-(6-purinyl)-l-lysine (3b)
antioxidant activity
This work was financially supported by University funds.
The ability of 3b to inhibit lipid peroxidation induced by a source
of free radicals such as tert-BOOH, was examined in rat liver micro-
somal membranes over 120 min of incubation. In order to evaluate
ferulate l-lysine and 6-MP antioxidant properties, the same exper-
iment was performed. The following graph (Fig. 3) shows the
complex 3b lipid peroxidation inhibition in relation to ferulate
lysine and 6-MP behaviour. It shows that 6-MP acts as a pro-oxidant
agent while ferulate l-lysine and its purinic thioester present a
strong antioxidant activity and a remarkable capacity to overthrow
the pro-oxidant effect of 6-MP itself. The association of 6-MP and
trans-ferulic acid has, therefore, a very important advantage: trans-
ferulic acid counteracts the pro-oxidant action of 6-MP and limits,
therefore, the side effects giving thus to our prodrug an important
References
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein–dye binding. Anal.
Biochem. 72, 248–254.
Cassano, R., Trombino, S., Cilea, A., Ferrarelli, T., Muzzalupo, R., Picci, N., 2009. l-
lysine pro-prodrug containing trans-ferulic acid for 5-aminosalicylic acid (5-
ASA) colon delivery: synthesis, characterization and in vitro antioxidant activity
evaluation. Chem. Pharm. Bull. 58, 103–105.
Evans, D.F., Pye, G., Bramley, R., Clark, A.G., Dyson, T.J., Hardcastle, J.D., 1988. Mea-
surement of gastrointestinal pH profiles in normal ambulant human subjects.
Gut 29, 1035–1041.
Freiberg, S., Zhu, X.X., 2004. Polymer microspheres for controlled drug release. Int.
J. Pharm. 282, 1–18.
Friend, D.R., 2005. New oral delivery systems for treatment of inflammatory bowel
disease. Adv. Drug Deliv. Rev. 57, 247–265.
Gazzaniga, A., Maroni, A., Sangalli, M.E., Zema, L., 2006. Time-controlled oral delivery
systems for colon targeting. Expert Opin. Drug Deliv. 3, 583–597.
Jain, A., Gupta, Y., Jain, S.K., 2007. Perspectives of biodegradable natural polysac-
charides for site-specific drug delivery to the colon. J. Pharm. Pharm. Sci. 10,
86–128.
Khan, M.Z.I., Prebeg, Z., Kurjakovic, N., 1999. A pH-dependent colon targeted oral
drug delivery system using methacrylic acid copolymers I. Manipulation of drug
release using Eudragit L100-55 and Eudragit S100 combinations. J. Contr. Rel. 58,
215–222.
Kinget, R., Kalala, W., Vervoort, L., van den Mooter, G., 1998. Colonic drug targeting.
J. Drug Target. 6, 129–149.
Koppel, D.E.J., 1972. Analysis of macromolecular polydispersity in inten-
sity correlation spectroscopy: the method of cumulants. Chem. Phys. 57,
4814–4820.
Makins, R.J., Cowan, R.E., 2001. 5-Amino-salicylate in the management of inflamma-
tory bowel disease. Colorectal. Dis. 3, 218–222.
Matthew, L., Maddess, M.N., Tackett, W.H., Brennan, P., Christopher, D.S., James, S.S.,
David, P.O., Steven, V.L., 2007. Total synthesis of rapamycin, natural products
synthesis. Angew. Chem. Int. 46, 591–597.
Ohta, T., Nakano, T., Egashira, Y., Sanada, H., 1997. Antioxidant activity of ferulic acid
beta-glucuronide in the LDL oxidation system. Biosci. Biotechnol. Biochem. 61,
1942–1943.
Pitarresi, G., Pierro, P., Giammona, G., Iemma, F., Muzzalupo, R., Picci, N., 2004.
Drug release from alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide-based
microparticles. Biomaterials. 25, 4333–4343.
Fig. 3. Evaluation of N␧-feruloyl-S-(6-purinyl)-l-lysine (3b) antioxidant activity.
(For interpretation of the references to color in this artwork, the reader is referred
to the web version of this article.)

296
S. Trombino et al. / International Journal of Pharmaceutics 420 (2011) 290–296
Provencher, S.W., 1982a. Constrained regularization method for inverting data rep-
resented by linear algebraic or integral equations. Comput. Phys. Commun. 27,
213–227.
Tatum, V.L., Changoit, C., Chow, C.K., 1990. Measurement of malondialdehyde by
high performance liquid chromatography with fluorescence detection. Lipids
25, 226–229.
Provencher, S.W., 1982b. Contin: a general purpose constrained regularization pro-
gram for inverting noisy linear algebraic and integral equations. Comput. Phys.
Commun. 27, 229–242.
Rubinstein, A., 2005. Colonic drug delivery. Drug Discov. Today Technol. 2, 33–37.
Sinha, V., Kumria, R., 2001. Colonic drug delivery: prodrug approach. Pharm. Res. 18,
557–562.
Taxonera, C., Schwartz, D.A., Olmo, D.G., 2009. Emerging treatments for complex
perianal fistula in Crohn’s. World J. Gastroenterol. 15, 4263–4272.
Trombino, S., Cassano, R., Bloise, E., Muzzalupo, R., Leta, S., Puoci, F., Picci, N., 2008.
Design and synthesis of cellulose derivatives with antioxidant activity. Macro-
mol. Biosci. 8, 86–95.
Van den Mooter, G., 2006. Colon drug delivery. Expert Opin. Drug Deliv. 3, 111–125.