Microwave synthesis and in vitro stability of diclofenac-β- cyclodextrin conjugate for colon delivery

Article abstract of DOI:10.1016/j.carbpol.2012.12.053

The aim of this work was to synthesize an ester prodrug of diclofenac and β-cyclodextrin suitable for colonic delivery. The synthesis of an ester linkage between diclofenac and β-cyclodextrin was conducted by the nucleophilic substitution of mono-6-tosyl-β-cyclodextrin under microwaves irradiation. After purification, the conjugate was characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry; infrared (IR) spectroscopy; proton nuclear magnetic resonance (¹H NMR) spectroscopy; and two-dimensional rotating frame nuclear overhauser effect (ROESY) spectroscopy. The purity was qualified by high pressure liquid chromatography (HPLC). To assess its potential for colonic delivery, the conjugate was evaluated for stability in simulated gastric and small intestinal fluids, and in fecal material from humans processed within a slurry under anaerobic conditions. The conjugate was successfully synthesized with a yield of 20% following purification. The mass spectra showed the parent peak m/z 1434 corresponding to [conjugate+Na] adduct. IR and NMR results confirmed that the carboxyl group of diclofenac is covalently bound to one of the hydroxyl groups of cyclodextrin by an ester linkage. Moreover, ROESY data indicated that the formation of the conjugate is not accompanied by the inclusion of diclofenac within the cyclodextrin. The conjugate was otherwise stable in simulated gastric and small intestinal conditions, but was also readily hydrolyzed liberating diclofenac in less than 2 h within the human fecal slurry. This confirmed the potential for this new prodrug as a carrier for colonic delivery.

Full text of DOI:10.1016/j.carbpol.2012.12.053

Carbohydrate Polymers 93 (2013) 512–517
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Microwave synthesis and in vitro stability of diclofenac-␤-cyclodextrin conjugate
for colon delivery
Amélia C.F. Vieiraa,b,d, Arménio C. Serrab, Rui A. Carvalhoc, Alexandra Gonsalvesb, Ana Figueirasa,
Francisco J. Veigaa, Abdul W. Basitd, António M. d’A. Rocha Gonsalvesb,∗
a Centre for Pharmaceutical Studies, Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Portugal
b Chymiotechnon and Department of Chemistry, Faculty of Science and Technology, University of Coimbra, Portugal
c Department of Life Sciences, Center for Neuroscience and Cell Biology, Faculty of Science and Technology, University of Coimbra, Portugal
d Department of Pharmaceutics, UCL School of Pharmacy, University College London, United Kingdom
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 was to synthesize an ester prodrug of diclofenac and ␤-cyclodextrin suitable for
colonic delivery. The synthesis of an ester linkage between diclofenac and ␤-cyclodextrin was con-
ducted by the nucleophilic substitution of mono-6-tosyl-␤-cyclodextrin under microwaves irradiation.
After purification, the conjugate was characterized by matrix-assisted laser desorption/ionization-time
of flight (MALDI-TOF) mass spectrometry; infrared (IR) spectroscopy; proton nuclear magnetic resonance
(1H NMR) spectroscopy; and two-dimensional rotating frame nuclear overhauser effect (ROESY) spec-
troscopy. The purity was qualified by high pressure liquid chromatography (HPLC). To assess its potential
for colonic delivery, the conjugate was evaluated for stability in simulated gastric and small intestinal
fluids, and in fecal material from humans processed within a slurry under anaerobic conditions. The con-
jugate was successfully synthesized with a yield of 20% following purification. The mass spectra showed
the parent peak m/z 1434 corresponding to [conjugate+Na] adduct. IR and NMR results confirmed that
the carboxyl group of diclofenac is covalently bound to one of the hydroxyl groups of cyclodextrin by an
ester linkage. Moreover, ROESY data indicated that the formation of the conjugate is not accompanied by
the inclusion of diclofenac within the cyclodextrin. The conjugate was otherwise stable in simulated gas-
tric and small intestinal conditions, but was also readily hydrolyzed liberating diclofenac in less than 2 h
within the human fecal slurry. This confirmed the potential for this new prodrug as a carrier for colonic
delivery.
Received 21 September 2012
Received in revised form
23 November 2012
Accepted 13 December 2012
Available online 7 January 2013
Keywords:
Large intestine
Colonic targeting
Microbiota
Fermentation
Prodrugs
Oligosaccharides
© 2013 Elsevier Ltd. All rights reserved.
1. Introduction
and associated enzymatic activity moving from the small intes-
tine to the colon (McConnell, Fadda, & Basit, 2008). For example,
Oral colon-specific drug delivery has progressively gained
importance for the localized delivery of therapeutic agents in the
treatment of disorders such as ulcerative colitis and Crohn’s dis-
ease. Additionally, colon-specific delivery provides an opportunity
for the systemic delivery of drugs utilized in the treatment of dis-
eases sensitive to circadian rhythms, including asthma, angina and
arthritis (McConnell, Liu, & Basit, 2009).
Though a number of approaches have been proposed for the
colonic delivery of therapeutic agents (Yang, Chu, & Fix, 2002),
the use of gastrointestinal microbiota as a prompt offers a viable
and attractive method of effecting drug release in the colon.
This approach exploits the abrupt increase in bacterial population
azo-bonded prodrugs of mesalazine – including sulfasalazine, bal-
salazide and olsalazine – are used clinically for the treatment of
ulcerative colitis. These prodrugs pass intact through the upper gas-
trointestinal tract, but once in the colon, the azo-bond is cleaved by
the resident microbiota, releasing the active drug molecule from
the carrier (Dhaneshwar & Vadnerkar, 2011).
The oligosaccharide ␤-cyclodextrin can function as a carrier for
colonic delivery.
␤-Cyclodextrin is a cyclic oligosaccharide derived from starch
consisting of seven glucose units linked by ␣-1,4 glycosidic bonds
in a doughnut-shaped form. As a consequence of its chemical struc-
ture, ␤-cyclodextrin can form inclusion complexes with smaller
molecules that fit into their cavities. ␤-Cyclodextrin is not absorbed
through biological membranes in the gastrointestinal tract, but it is
fermented into small saccharides by the microbiota of the colon – in
particular, Bacteroides species (Chourasia & Jain, 2003; Flourié et al.,
1993; Park et al., 2000; Sinha & Kumria, 2001). Active molecules can
∗
Corresponding author at: Apartado 3096, Rua Larga 3004 535 Coimbra, Portugal.
Tel.: +351 239 104191; fax: +351 239 827 703.
E-mail address: arg@qui.uc.pt (A.M.d’A. Rocha Gonsalves).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2012.12.053

A.C.F. Vieira et al. / Carbohydrate Polymers 93 (2013) 512–517
513
be attached covalently to the primary or secondary hydroxyl groups
of ␤-cyclodextrin producing prodrugs with the ability to remain
intact in the upper gastrointestinal tract. Once such molecules
reach the microbiota of the colon, they are subsequently cleaved
to release the active drug (Uekama, 2004).
Moreover, fermentation of ␤-cyclodextrin leads to production
of short-chain fatty acids (SCFA) that can contribute to the mainte-
nance of the health and integrity of the colonic epithelium (Giardina
& Inan, 1998). In this respect, therefore, the use of ␤-cyclodextrin
as a carrier provides considerable health benefits and is addition-
ally ‘Generally Regarded As Safe’ (GRAS) (Astray, Gonzalez-Barreiro,
Mejuto, Rial-Otero, & Simal-Gándara, 2009).
Diclofenac [2-(2,6-dicloranilino) phenylacetic acid] is a non-
steroidal anti-inflammatory drug, and much interest surrounds
the notion of targeting its release in the colon, given that such
an approach would allow for circumvention of the adverse gas-
tric effects notoriously associated with drugs of this class. It is
also widely believed that the colonic delivery of diclofenac would
provide a suitable tactic for the management of arthritis pain by
chronotherapy (Lin & Kawashima, 2012). Furthermore, diclofenac
is known to be well-absorbed in the colon (Gleiter et al., 1985),
and has demonstrated an effective chemopreventive action in colon
cancer (Jasmeet & Nath, 2010; Saini, Kaur, Sharma, & Sanyal, 2009).
In this present study, we have investigated a method to prepare
diclofenac-␤-cyclodextrin prodrug suitable for site-specific deliv-
ery in the colon. The preparation method involves modification of
a ␤-cyclodextrin hydroxyl group by an electrophylic reagent fol-
lowed by nucleophylic substitution under microwaves irradiation.
The stability of the conjugate was determined in simulated stomach
and small intestinal fluids, and in human fecal slurry to mimic the
conditions of the colon, to determine the suitability of the conjugate
to release diclofenac in the colon.
An HPLC system (model HP1100 series, Hewlett Packard,
Germany) equipped with an autosampler (Agilent 1100 series,
Germany) was used. A reversed-phase X-Terra C-18 column,
5 ␮m, 4.6 mm × 250 mm (Waters, USA), with a pre-column, was
employed. Mobile phase consisted of acetonitrile and 0.4% trifluo-
roacetic acid (TFA) in water with a flow rate 1.0 mL/min, an injection
volume of 20 ␮L and detection at 254 nm at 30 ◦C.
2.3. Synthesis of diclofenac-ˇ-cyclodextrin conjugate
Diclofenac-␤-cyclodextrin was synthesized through a 2-step
process involving tosylation of ␤-cyclodextrin (step 1) and
nucleophylic substitution of tosylated ␤-cyclodextrin by sodium
diclofenac under microwaves (step 2).
Step 1: Tosylated ␤-cyclodextrin was synthesized adapting the
procedure described by McNaughton, Engman, Birmingham, Powis,
and Cotgreave (2004). Briefly, to a solution of 5 g of ␤-cyclodextrin
(4.4 mmol) in water (110 mL) 1.25 g of p-toluenesulfonyl chloride
(6.55 mmol) was added and the resultant solution was stirred
at room temperature for 2 h under inert atmosphere. Aqueous
NaOH (2.5 M, 20 mL) was added and the solution stirred for 10 min
before unreacted p-toluenesulfonyl chloride was filtered off. 5.8 g
of ammonium chloride (108 mmol) was added to lower the pH to
approximately pH 8. The solution was cooled overnight and the
resultant white precipitate collected by filtration. The white pow-
der was washed with acetone and water to remove the non-reacted
cyclodextrin and then dried under vacuum. The product obtained
is used directly in the next step.
Step 2: Sodium diclofenac (0.333 mmol) was dissolved in 3 mL of
anhydrous DMF containing ␤-cyclodextrin tosylate (0.327 mmol)
in an appropriate thick-walled glass vial. The reaction vessel was
then sealed with a teflon cap and the reaction mixture magnetically
stirred and heated at 140 ◦C for 40 min under focused microwave
irradiation with an initial power setting of 75 W (CEM Discover
S-class single mode microwave reactor). The product obtained
was precipitated in acetone, filtered, washed several times with
acetone and ethyl ether and dried. In order to obtain a puri-
fied sample 300 mg of the crude product was passed through
DIAION HP-20 ion-exchange chromatography column eluting with
water/methanol, and steadily increasing the methanol content. The
conjugate was eluted with 80% methanol. Methanol in the elu-
ate was removed under reduced pressure, the solution lyophilized
in a freeze-dryer (Lyph-lock 6 apparatus, Labconco) for 72 h
and the diclofenac-␤-cyclodextrin was obtained with a yield of
20%.
2. Materials and methods
2.1. Chemicals
␤-Cyclodextrin hydrate, p-toluenesulfonyl chloride, DIAION HP-
20 were obtained from Sigma. Sodium diclofenac was kindly
provided by Labesfal Genéricos. Dimethylformamide (DMF) was
purchased from Fisher Scientific. Other chemicals and solvents
were of analytical reagent grade and deionized water was used.
2.2. Characterization
Thin layer chromatography (TLC) was conducted on aluminum
plates percoated with silica gel F254 (Merck & Co.) and eluted with
a mixture of 2-propanol/ethyl acetate/water/ammonia (6:1:3:1) or
acetonitrile/water/ammonia (6:3:1).
1H NMR (500 MHz, DMSO-d6) ı (ppm) 7.52 (d, H-5ꢀ, H-3ꢀ of
Diclofenac), 7.22 (d, H-4ꢀꢀ of Diclofenac), 7.20 (t, H-4ꢀ of Diclofenac),
7.05 (t, H-6ꢀꢀ of Diclofenac), 6.83 (t, H-5ꢀꢀ of Diclofenac), 6.25 (d, H-
7ꢀꢀ of Diclofenac), 5.66–5.84 (m, OH-2 and OH-3 of ␤-cyclodextrin),
4.83 (d, H-1 of ␤-cyclodextrin), 4.21–4.55 (m, OH-6, H-6 of ␤-
cyclodextrin), 3.50–3.90 (m, H-6, H-3 and H-5 of ␤-cyclodextrin),
3.25–3.43 (m, H-2, H-4 of ␤-cyclodextrin), MS (MALDI) m/z for
C56H79Cl2NNaO36, found 1434.308 [conjugate+Na].
For spot detection the plates were immersed in
a mix-
ture of p-anisaldehyde (2 mL)/ethanol (36 mL)/acetic acid (5–6
drops)/sulfuric acid (2 mL). The plate was heated to 150 ◦C for 5 min
in order to visualize the spots.
The identification of the obtained compounds was confirmed by
one or more of the following techniques: proton nuclear magnetic
resonance (1H NMR) spectroscopy; matrix-assisted laser desorp-
tion/ionization (MALDI) spectroscopy; infrared (IR) spectroscopy;
and high performance liquid chromatography (HPLC).
2.4. Stability of diclofenac-ˇ-cyclodextrin in simulated gastric
and small intestinal fluids.
Simulated gastric fluid (SGF) was prepared according to USP
specifications. Sodium chloride (200 mg) was added to a 100 mL
flask and dissolved in water followed by addition of 700 ␮L HCl
(10 M) to adjust the pH to 1.2. Pepsin (320 mg) was then added
to the medium. The simulated small intestinal fluid (SIF) was also
prepared according to USP specifications through dissolution of
680 mg monobasic potassium phosphate in water. To this solu-
tion 7.7 mL of 0.2 M sodium hydroxide solution was added and
the remaining water added to make the volume up to 100 mL.
1H NMR spectra were acquired on a Varian Unity-500MHz
spectrometer, using deuterated dimethyl sulfoxide (DMSO-d6) as
solvent and TMS was used as an internal reference.
MALDI-TOF mass spectra were obtained on an Autoflex III,
Bruker using methanol and water as solvents and ␣-cyano-4-
hydroxy-cinnamic acid (HCCA) as matrix.
IR spectra was obtained using Thermo Scientific Nicolet 6700
FT-IR spectrometer by scanning KBr discs of the samples.

514
A.C.F. Vieira et al. / Carbohydrate Polymers 93 (2013) 512–517
After adjusting the pH to 6.8, 1 g pancreatin (from porcine pan-
creas, 3USP units activity/g) was added and shaken gently until
dissolved.
In contrast to these initial approaches, we succeeded in
synthesizing of the diclofenac-␤-cyclodextrin conjugate by the
nucleophylic substitution of a 6-tosyl group under microwave
radiation. The major challenge of this first step was to achieve
monotosylation of cyclodextrin specifically to the 6-position
without considerable amounts of primary and secondary side
multi-tosylated by-products (Brady, Lynam, O‘Sullivan, Ahern, &
Darcy, 2000; Khan, Forgo, Stine, & D’Souza, 1998). This selec-
tive sulfonylation generally occurs in dry pyridine (Tang & Ng,
2008), or in water at alkaline pH (Brady et al., 2000). Pyridine
is a non-user-friendly solvent and forms a pyridinium complex
with the cyclodextrin cavity, complicating the process of purifica-
tion (Khan et al., 1998). Consequently, the method using deionized
water as a solvent was followed in order to prepare the 6-
p-toluenosulfonyl-␤-cyclodextrin derivative (Brady et al., 2000).
Following the procedure as described above we obtained gram
amounts of the 6-p-toluenosulfonyl-␤-cyclodextrin that was char-
acterized by NMR spectroscopy and MALDI-TOF spectrometry. For
the substitution reaction, this product was used without purifica-
tion, despite the presence of small amounts of di- and tri-tosilated
derivatives as verified by MALDI analysis.
Microwave irradiation has long been viewed as an interesting
alternative to the use of classical heating systems, and partic-
ularly in the field of organic synthesis, largely due to frequent
increases in reaction rates, improved yields and selectivities asso-
ciated with this technique (Richel, Laurent, Wathelet, Wathelet, &
Paquot, 2011).
Nucleophylic substitution of tosyl group by the sodium
diclofenac was achieved in DMF under microwave irradiation. The
diclofenac-␤-cyclodextrin conjugate was obtained after a system-
atic study of temperature and microwave conditions.
Aqueous solutions of diclofenac-␤-cyclodextrin conjugate
0.8 mg/mL were prepared in SGF and in SIF. To guarantee the
complete dissolution of the conjugate in the medium 1% of DMF
was used. These solutions were incubated at 37 ◦C and shaken at
100 rpm. At fixed intervals, 100 ␮L of fluid was withdrawn, mixed
with 400 ␮L of methanol and centrifuged at 10,000 rpm for 10 min
at room temperature. The supernatant was then removed and ana-
lyzed via HPLC to determine the concentration of conjugate. Each
experiment was performed in triplicate.
2.5. Stability of diclofenac-ˇ-cyclodextrin in human fecal slurry
Fecal slurries were utilized to simulate the conditions of the
colon. Fresh feces were collected from healthy adults and trans-
ferred into an anaerobic workstation at 37 ◦C and relative air
humidity of 70% as soon as possible after defecation. The feces were
diluted with phosphate buffer saline (British Pharmacopeia) pH 6.8
in order to obtain a 40% w/w slurry. The mixture was then homog-
enized and sieved through an open mesh fabric (Sefar NitexTM,
pore size 350 ␮m) to remove any unhomogenised fibrous mate-
rial. Afterwards, the sieved homogenized fecal slurry was diluted
50% (w/w) with basal medium containing peptone water, yeast
extract, NaCl, K2HPO4, MgSO4·7H2O, CaCl2·6H2O, NaHCO3, Haemin,
l-cysteine HCl, bile salts, tween 80, vitamin K and resazurin (Basit
& Lacey, 2001).
Solutions of diclofenac-␤-cyclodextrin (2.4 mg/mL) were pre-
pared in PBS 6.8 (British Pharmacopeia) with DMF. 300 ␮L of this
solution was mixed with 900 ␮L of fecal slurry in the anaerobic
workstation. The final concentrations of the conjugate and DMF
were 0.6 mg/mL and 1.0%, respectively. A control experiment was
also run in parallel in fecal slurry that was subjected to autoclaving
at 130 ◦C for 20 min. This was conducted to inactivate the bacterial
enzymes in the slurry.
An HPLC study of different samples prepared under different
reaction temperatures showed that the use of 140 ◦C produced the
best yield. As temperature increases, it is also possible to observe
a decrease in diclofenac-␤-cyclodextrin yields. This is likely due to
the degradation of diclofenac at high temperatures (Galmier et al.,
2005; Tudja et al., 2001). The use of microwave radiation addition-
ally led to the conjugate formation, with a short reaction time and
less solvent comparatively to conventional processes required for
the preparation of cyclodextrin conjugates (Hirayama et al., 2000).
After chromatographic purification, about 20% yield of product was
achieved, and the microwave approach to the synthesis of this con-
jugate proved to be consistently reproducible.
Thereafter, these solutions were incubated and shaken at
100 rpm. At fixed intervals, 100 ␮L of fluid was withdrawn, mixed
with 400 ␮L of methanol and centrifuged at 10,000 rpm for 10 min
at room temperature. The supernatant was then removed and ana-
lyzed by HPLC. Each experiment was performed in triplicate.
The chemical structure of the conjugate was confirmed and
characterized by IR, 1H NMR, ROESY spectroscopy and MALDI-TOF
spectrometry, and the purity confirmed by HPLC.
3. Results and discussion
3.1. Synthesis of diclofenac-ˇ-cyclodextrin conjugate
Initially, various approaches were investigated to synthesize
the cyclodextrin conjugate. These included: nucleophylic sub-
stitution with conventional heating (Hirayama et al., 2000);
activation of diclofenac using carbodiimides (Dev, Mhaske, Kadam,
& Dhaneshwar, 2007; Udoa et al., 2010), and the formation of an
acid chloride (Cassano et al., 2010; Ventura et al., 2003). All three
approaches were unsuccessful in the synthesis of the conjugate.
We came to the conclusion that these approaches did not work
due to the particular structure of diclofenac since these strate-
gies had success in other cyclodextrin conjugates. As support to
this conclusion the work of Roy et al. (2001) and also the work
of Tudja, Khan, Mestrovic, Horvat, and Golja (2001) showed that
under heating conditions diclofenac can suffer dehydratation and
an intramolecular cyclization. In other case where an activation of
the carboxylic acid was attempted we suggest that nucleophylic
attack of the hydroxyl group of cyclodextrin cannot compete with
the more favorable intramolecular acylation by the nearby amino
group.
3.2. Characterization of diclofenac-ˇ-cyclodextrin
Mass spectrum (MALDI-TOF) of the product shows only a signal
m/z of 1434.308 corresponding to the [M+Na] adduct. No other rel-
evant mass signal was observed as shown in Fig. 1. The formation of
the conjugate was proved by comparison of the 1H NMR spectrum
of the free ␤-cyclodextrin (Fig. 2II-A); and of the sodium diclofenac
(Fig. 2II-B); with the spectrum of the diclofenac-␤-cyclodextrin
conjugate (Fig. 2II-C). The ester bond occurs at the hydroxyl of the 6-
position of cyclodextrin once the protons of cyclodextrin secondary
hydroxyl groups do not demonstrate a significant shift when com-
pared to the dramatic upfield shift observed in the protons of
primary hydroxyl (change from 4.60 ppm to 4.21–4.55 ppm). More-
over, the 6-hydroxyl substitution is also confirmed by the reduction
in the integration of the peak due to the hydroxyl protons in the 6-
position. The H-6 protons linked to diclofenac resonate downfield
relative to the unsubstituted H-6 protons, and present a distinct
multiplicity.

A.C.F. Vieira et al. / Carbohydrate Polymers 93 (2013) 512–517
515
Fig. 1. MALDI mass spectrum of diclofenac-␤-cyclodextrin conjugate.
ROESY experiments shown in Fig. 3 indicate that the only inter-
molecular correlations observed were between the H-4ꢀꢀ and H-6ꢀꢀ
protons of the phenyl acetate ring and the H-6 protons of the
␤-cyclodextrin, corroborating the hypothesis of diclofenac non-
penetration into the hydrophobic core of cyclodextrin.
Fig. 3. 1H NMR ROESY spectra of the diclofenac-␤-conjugate.
IR spectroscopy also aided the identification of the diclofenac-
␤-cyclodextrin conjugate (see Fig. 4) (Hamdi, Abderrahim, &
Meganem, 2010; Jing, Yanping, Baoguo, & Chengtao, 2011; Özkan,
Atay, Dïkmen, Is¸ imer, & Aboul-Enein, 2000; Wang, Han, Feng, &
Pang, 2006). The spectrum of the conjugate (Fig. 4C) shows a pattern
distinct from the spectrum of ␤-cyclodextrin (Fig. 4B) and of the
3.3. Stability studies of diclofenac-ˇ-cyclodextrin in simulated
gastric and small intestinal fluids and in human fecal slurry
Diclofenac-␤-cyclodextrin must pass intact through the stom-
ach and the small intestine, and reach the colon where it will act
upon enzymes of the colonic microbiota to release the active drug.
Thus, in order to determine the stability of the conjugate in the
stomach and small intestine, simulated gastric and small intesti-
nal fluids were used. Results from these in vitro hydrolysis studies,
as shown in Fig. 5, revealed that the conjugate is stable in these
simulated fluids.
diclofenac (Fig. 4A). The broad band at 3300–3500 cm−1 due to O
H
stretching is narrower and more closely resembles the correspond-
ing ␤-cyclodextrin. Equally, the region at 1000–1200 cm−1 due to
C
O C stretching is similar in spectrum (B), but with a new promi-
nent band located at 1028 cm−1. Near the H
O H bending band of
␤-cyclodextrin at 1650 cm−1, a new band appears at 1729 cm−1 due
to the created ester bond.
Fig. 2. I-Structure of the diclofenac-␤-cyclodextrin; II-A-1H NMR spectrum of hydrated ␤-cyclodextrin hydrated. II-B-1H NMR spectrum of sodium diclofenac. III-C-1H NMR
spectrum of diclofenac-␤-cyclodextrin conjugate.

516
A.C.F. Vieira et al. / Carbohydrate Polymers 93 (2013) 512–517
Fig. 4. IR spectra of diclofenac (A), ␤-cyclodextrin (B) and diclofenac-␤-diclofenac conjugate (C).
The lack of a Pharmacopeia simulated colonic fluid for in vitro
diclofenac (Fig. 6). By contrast, the conjugate was stable in the auto-
claved fecal slurry (control). These results confirm that cleavage of
the conjugate is due to bacterial enzymatic activity in the slurry.
Moreover, release of the drug from the prodrug depends not only
of the hydrolysis of the ester linkage but also of the integrity of the
cyclodextrin, since release only takes place where cyclodextrin are
fermented into small saccharides by the colonic microbiota (Flourié
et al., 1993).
studies warrants the use of other methodology to mimic the envi-
ronment of the human colon, in this case, the use of fresh human
feces within a suitable medium has been identified as a suitable
alternative (Sousa et al., 2008). The conjugate was added to the
slurry and the results demonstrated that the conjugate was readily
hydrolyzed in the human fecal slurry, and within 2 h the conjugate
is completely degraded, as evidenced by the rapid liberation of free
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
60
120
180
0
60
120
180
240
Time (minutes)
Time (minutes)
Fig. 6. Mean levels of diclofenac-␤-cyclodextrin (ꢀ) and diclofenac (ꢁ) in human
fecal slurry (test). Mean levels of diclofenac-␤-cyclodextrin (ꢁ) and diclofenac (᭹)
in autoclaved fecal slurry (control). Each point represents mean S.D. (n = 3).
Fig. 5. Mean levels of diclofenac-␤-cyclodextrin conjugate in simulated gastric (ꢀ)
and small intestinal (ꢁ) fluids. Each point represents mean S.D. (n = 3).

A.C.F. Vieira et al. / Carbohydrate Polymers 93 (2013) 512–517
517
4. Conclusion
Jasmeet, K., & Nath, S. S. (2010). Induction of apoptosis as a potential chemo-
preventive effect of dual cycloxygenase inhibitor, diclofenac, in early colon
carcinogenesis. Journal of Environmental Pathology, Toxicology and Oncology, 29,
41–53.
Jing, W., Yanping, C., Baoguo, S., & Chengtao, W. (2011). Physicochemical and
release characterisation of garlic oil-[beta]-cyclodextrin inclusion complexes.
Food Chemistry, 127, 1680–1685.
Khan, A. R., Forgo, P., Stine, K. J., & D’Souza, V. T. (1998). Methods for selective
modifications of cyclodextrins. Chemical Reviews, 98, 1977–1996.
Lin, S.-Y., & Kawashima, Y. (2012). Current status and approaches to developing
press-coated chronodelivery drug systems. Journal of Controlled Release, 157,
331–353.
McConnell, E. L., Fadda, H. M., & Basit, A. W. (2008). Gut instincts: Explorations in
intestinal physiology and drug delivery. International Journal of Pharmaceutics,
364, 213–226.
In this work, synthesis of diclofenac-␤-cyclodextrin conjugate
was made possible with the use of microwave irradiation as a
principal energy source. After synthesis, this prodrug was fully
characterized and the purity verified by HPLC. Stability studies
revealed that the conjugate is able to target the colon, given that
it is completely stable in simulated gastric and in simulated small
intestinal fluids but is degraded in the fecal slurry. This stability
behavior is due to specific hydrolysis of ␤-cyclodextrin by colonic
microbiota which was probed using the fecal slurry system. These
preliminary results are promising, and suggest that the prodrug is
capable of facilitating release of the active, diclofenac, in the colon.
McConnell, E. L., Liu, F., & Basit, A. W. (2009). Colonic treatments and targets: Issues
and opportunities. Journal of Drug Targeting, 17, 335–363.
McNaughton, M., Engman, L., Birmingham, A., Powis, G., & Cotgreave, I. A. (2004).
Cyclodextrin-derived diorganyl tellurides as glutathione peroxidase mimics and
inhibitors of thioredoxin reductase and cancer cell growth. Journal of Medicinal
Chemistry, 47, 233–239.
Acknowledgment
Özkan, Y., Atay, T., Dïkmen, N., Is¸ imer, A., & Aboul-Enein, H. Y. (2000). Improvement
of water solubility and in vitro dissolution rate of gliclazide by complexation
with [beta]-cyclodextrin. Pharmaceutica Acta Helvetiae, 74, 365–370.
Park, K.-H., Kim, T.-J., Cheong, T.-K., Kim, J.-W., Oh, B.-H., & Svensson, B. (2000). Struc-
ture, specificity and function of cyclomaltodextrinase, a multispecific enzyme
of the alpha-amylase family. Biochimica et Biophysica Acta, 1478, 165–185.
Richel, A., Laurent, P., Wathelet, B., Wathelet, J.-P., & Paquot, M. (2011). Microwave-
assisted conversion of carbohydrates. State of the art and outlook. Comptes
Rendus de Chimica, 14, 224–234.
Roy, J., Islam, M., Khan, A. H., Das, S. C., Akhteruzzaman, M., Deb, A. K., et al. (2001).
Diclofenac sodium injection sterilized by autoclave and the occurrence of cyclic
reaction producing a small amount of impurity. Journal of Pharmaceutical Sci-
ences, 90, 541–544.
Saini, M. K., Kaur, J., Sharma, P., & Sanyal, S. N. (2009). Chemopreventive response
of diclofenac, a non-steroidal anti-inflammatory drug in experimental carcino-
genesis. Nutricion Hospitalaria, 24, 717–723.
Sinha, V. R., & Kumria, R. (2001). Polysaccharides in colon specific drug delivery.
International Journal of Pharmaceutics, 224, 19–38.
Sousa, T., Paterson, R., Moore, V., Carlsson, A., Abrahamsson, B., & Basit, A. W. (2008).
The gastrointestinal microbiota as a site for the biotransformation of drugs.
International Journal of Pharmaceutics, 363, 1–25.
Tang, W., & Ng, S.-C. (2008). Facile synthesis of mono-6-amino-6-deoxy-␣-, ␤-, ␥-
cyclodextrin hydrochlorides for molecular recognition, chiral separation and
drug delivery. Nature Protocols, 3, 691–697.
The authors thank the Fundac¸ ão para a Ciência e Tecnologia,
Portugal, for their financial support (SFRH/BD/44925/2008).
References
Astray, G., Gonzalez-Barreiro, C., Mejuto, J. C., Rial-Otero, R., & Simal-Gándara, J.
(2009). A review on the use of cyclodextrins in foods. Food Hydrocolloids, 23,
1631–1640.
Basit, A. W., & Lacey, L. F. (2001). Colonic metabolism of ranitidine: Implications for
its delivery and absorption. International Journal of Pharmaceutics, 227, 157–165.
Brady, B., Lynam, N., O’Sullivan, T., Ahern, C.,
& Darcy, R. (2000). 6A-O-p-
toluenosulfonyl-␤-cyclodextrin. Organic Syntheses, 77, 220.
Cassano, R., Trombino, S., Ferrarelli, T., Barone, E., Arena, V., Mancuso, C., et al. (2010).
Synthesis, characterization, and anti-inflammatory activity of diclofenac-bound
cotton fibers. Biomacromolecules, 11, 1716–1720.
Chourasia, M. K., & Jain, S. K. (2003). Pharmaceutical approaches to colon targeted
drug delivery systems. Journal of Pharmaceutical Sciences, 6, 33–66.
Dev, S., Mhaske, D. V., Kadam, S., & Dhaneshwar, S. (2007). Synthesis and pharmaco-
logical evaluation of cyclodextrin conjugate prodrug of mefenamic acid. Indian
Journal of Pharmaceutical Sciences, 69, 69–72.
Dhaneshwar, S. S., & Vadnerkar, G. (2011). Rational design and development of
colon-specific prodrugs. Current Topics in Medicinal Chemistry, 11, 2318–2345.
Flourié, B., Molis, C., Achour, L., Dupas, H., Hatat, C., & Rambaud, J. C. (1993). Fate of
beta-cyclodextrinin the human intestine. Journal of Nutrition, 123, 676–680.
Galmier, M.-J., Bouchon, B., Madelmont, J.-C., Mercier, F., Pilotaz, F., & Lartigue, C.
(2005). Identification of degradation products of diclofenac by electrospray ion
trap mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 38,
790–796.
Giardina, C., & Inan, M. S. (1998). Nonsteroidal anti-inflammatory drugs, short-chain
fatty acids, and reactive oxygen metabolism in human colorectal cancer cells.
Biochimica et Biophysica Acta, 1401, 277–288.
Gleiter, C. H., Antonin, K.-H., Bieck, P., Godbillon, J., Schönleber, W., & Malchow, H.
(1985). Colonoscopy in the investigation of drug absorption in healthy volun-
teers. Gastrointestinal Endoscopy, 32, 71–73.
Hamdi, H., Abderrahim, R., & Meganem, F. (2010). Spectroscopic studies of inclusion
complex of [beta]-cyclodextrin and benzidine diammonium dipicrate. Spec-
trochimica Acta, Part A, 75, 32–36.
Hirayama, F., Ogata, T., Yano, H., Arima, H., Udo, K., Takano, M., et al. (2000). Release
characteristics of a short-chain fatty acid, n-butyric acid, from its ␤-cyclodextrin
ester conjugate in rat biological media. Journal of Pharmaceutical Sciences, 89,
1486–1495.
Tudja, P., Khan, M. Z. I., Mestrovic, E., Horvat, M., & Golja, P. (2001). Thermal behaviour
of diclofenac sodium: Decomposition and melting characteristics. Chemical and
Pharmaceutical Bulletin, 49, 1245–1250.
Udoa, K., Hokonoharaa, K., Motoyamaa, K., Arimaa, H., Hirayamab, F., & Uekamab,
K. (2010). 5-Fluorouracil acetic acid/␤-cyclodextrin conjugates: Drug release
behavior in enzymatic and rat cecal media. International Journal of Pharmaceutics,
388, 95–100.
Uekama, K. (2004). Design and evaluation of cyclodextrin-based drug formulation.
Chemical and Pharmaceutical Bulletin, 52, 900–915.
Ventura, C. A., Paolino, D., Pedotti, S., Pistarà, V., Corsaro, A.,
& Puglisi, G.
(2003). Synthesis, characterization and in vitro evaluation of dimethyl-beta-
cyclodextrin-4-biphenylylacetic acid conjugate. Journal of Drug Targeting, 11,
233–240.
Wang, H. Y., Han, J., Feng, X. G., & Pang, Y. L. (2006). Study of inclusion complex for-
mation between tropaeolin OO and [beta]-cyclodextrin by spectrophotometry
and Infrared spectroscopy. Spectrochimica Acta, Part A, 65, 100–105.
Yang, L., Chu, J. S., & Fix, J. A. (2002). Colon-specific drug delivery: New approaches
and in vitro/in vivo evaluation. International Journal of Pharmaceutics, 235, 1–15.