Journal of Controlled Release 88 (2003) 19

www.elsevier.com / locate / jconrel

Dendrimers conjugates for colonic delivery of 5-aminosalicylic

acid
Ruedeekorn Wiwattanapatapee a , *, Luelak Lomlim b , Krisanee Saramunee b
a
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai,
Songkla 90112, Thailand
b
Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai,
Songkla 90112, Thailand

Received 15 May 2002; accepted 13 September 2002

Abstract

The water-soluble polyamidoamine (PAMAM) dendrimer conjugates for colonic delivery of 5-aminosalicylic acid
(5-ASA) were designed. The drug was bound to the dendrimer using two different spacers containing azo-bond,
p-aminobenzoic acid (PABA) and p-aminohippuric acid (PAH). Incubation of PAMAM dendrimer conjugates containing
PABA and PAH spacers with rat cecal contents at 37 8C gradually released 5-ASA with time and the amount of drug released
was 45.6 and 57.0% of the dose in 24 h, respectively. The release of the drug from the commercial prodrug, sulfasalazine
was significantly faster than both conjugates (80.2% of the dose in 6 h). No 5-ASA was detected from the incubation of
dendrimer conjugates with the homogenate of the stomach or phosphate buffer, pH 1.2 and 6.8. Only a small amount of
5-ASA was found after incubation of both conjugates with the homogenate of the small intestine for 12 h. It appears that this
PAMAM dendrimer can be developed for use as a carrier for colon-specific drug delivery.
2002 Elsevier Science B.V. All rights reserved.

Keywords: Dendrimers; Colon-specific drug delivery; 5-Aminosalicylic acid

1. Introduction and high residence time compared to the stomach

Delivery of orally administered drugs specifically
to the colon has a number of important implications
in the field of pharmacotherapy. Diseases of the
colon such as irritable bowel syndrome, Crohns
disease and ulcerative colitis are effectively treated
when the anti-inflammatory agents are applied direct-
ly to the affected area. Recently, colon targeting
becomes much attractive for delivery of peptides and
proteins due to the low digestive enzymatic activity
*Corresponding author.
and small intestine [16]. In addition, colonic deliv-
ery of drugs may also be useful when intentional
delayed drug absorption is required such as asthma,
gastric ulcer or arthritis [7].
Several approaches utilized in achieving colon
targeting include use of pH-sensitive polymer coat-
ings [8,9], time-dependent formulations [10], bacteri-
al degradable coatings [1115], biodegradable poly-
mer matrices and hydrogels [16,17] and prodrugs
[1821]. To date, many low molecular and macro-
molecular prodrugs have been prepared for colonic
delivery. A well-known commercial prodrug, sul-

0168-3659 / 02 / $ see front matter 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0168-3659(02)00461-3
2 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19
fasalazine is a 5-ASA prodrug which is reduced by
the colonic bacteria into 5-ASA and sulfapyridine.
5-ASA is largely unabsorbed from the colon and acts
as a topical anti-inflammatory. In contrast, sul-
fapyridine is well absorbed giving rise to side-effects
and toxicity, and as many as 30% of patients are
unable to tolerate treatment with sulfasalazine [22].
With the knowledge that the adverse effects associ-
ated with sulfasalazine are due to sulfapyridine,
many investigations started for the choice of a
suitable carrier for 5-ASA with minimum adverse
effects. Recently, polymeric prodrugs of 5-ASA
susceptible to azoreductase were developed. In these
prodrugs 5-ASA was linked to a non-absorbable
polymer, for example N-(2-hydroxy-
propyl)methacrylamide [20,21], polysul-
fonamidoethylene [23] and dextran [24,25]. Studies
showed that these prodrugs had good potential to act
as carriers for colon-specific drug delivery.
This article reports the rational design of den-
drimer conjugates to achieve the colonic delivery of
5-ASA, and evaluation of drug release from these
macromolecular prodrugs. Dendrimers have a de-
fined structure with a large number of functional
groups at their multiple chain ends. This type of
structure allows conjugation of one molecule carrier
to many drug molecules via designed linkages [26].
Previously, biocompatibility of polyamidoamine
(PAMAM) dendrimers and their potential for use as
oral drug delivery systems have been reported
[27,28]. After oral administration in rat, the systemic
absorption of PAMAM dendrimer was very low or
negligible [29]. PAMAM dendrimers have thus be-
come an attractive option for use as drug carriers to
colon. In this study, the conjugates of PAMAM
dendrimer generation 3 (G3) with 32 amino terminal
groups and 5-ASA pro-moiety containing two differ-
ent azo linkers PABA or PAH were synthesized (Fig.
1). The release of drug from the conjugates in vitro
compared to sulfasalazine was also determined.
2. Materials and methods
2.1. Materials
PAMAM dendrimer generation 3 was obtained as
methanolic solutions (20 wt%) from Aldrich (USA).
Sulfasalazine and carbonyl di-imidazole (CDI) were
purchased from Fluka (Switzerland). Salicylic acid
(SA) and p-aminobenzoic acid (PABA) were from
Merck (Germany) and p-aminohippuric acid (PAH)
were from Sigma (USA). All other reagents and
solvents were of commercial products of analytical
or reagent grade. Wistar rats were from Prince of
Songkla University animal house.
2.2. Instrumentation
IR spectra were recorded by the use of KBr pellets
on a Perkin-Elmer FT-IR model 1600 spec-
trophotometer. NMR spectra were recorded by Var-
ian Nuclear Magnetic Resonance Spectrometer (500
MHz). UV spectra were obtained from Hewlett-Pac-
kard 8452 UVvisible spectrophotometer. High-per-
formance liquid chromatography (HPLC) was car-
ried out using the Waters Model. Spectra / Por
MWCO 2000 was used as the dialysis membrane.
2.3. Synthesis
2.3.1. Synthesis of PAMAMPABASA conjugates
Methanolic PAMAM dendrimer solution (equiva-
lent to PAMAM 0.025 mmol) was first dried under a
stream of nitrogen to give a solid residue. Solutions
of PABA (1.6 mmol) in 5 ml dimethylsulfoxide
(DMSO) and CDI (1.6 mmol) in 1.25 ml DMSO
were added. The mixture was stirred at room tem-
perature for 24 h. The PAMAMPABA conjugate
was purified by dialysis in distilled water (24 h). The
product was freeze-dried to obtain PAMAMPABA
conjugate as white solid. A solution of PAMAM
PABA conjugate in 4 ml water and a solution of
NaNO 2 (6.4 mmol) in 4 ml water were mixed. The
mixture was stirred in the ice bath (35 8C) for 15
min and acidified with 3 M HCl. The solution was
stirred for a further 5 min, and then tested for the
presence of excess nitrous acid using potassium
iodide / starch indicator paper. A solution of salicylic
acid (SA, 6.4 mmol) in 20 ml of 0.5 M NaOH was
stirred at 35 8C. The diazonium salt solution was
added to this solution and pH was then adjusted to
10 using 1 M NaOH. The reddish brown solution
was stirred for 30 min. The PAMAMPABASA
conjugate was purified by dialysis, and freeze-dried
to obtain reddish brown solid (69.4 mg, 35.6%
 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19 3

Fig. 1. Structures of PAMAM dendrimer G3 (A), PAMAMPABASA conjugate (B), and PAMAMPAHSA conjugate (C).
4 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19
yield). 1 H NMR spectrum (D 2 O): d 2.023.44 (484
H, multiplet, CH protons of PAMAM), 6.886.94
(1H, multiplet, aromatic proton of SA), 7.547.68
(2H, doublet, aromatic protons of PABA) 7.667.80
(1H, doublet, aromatic proton of SA), 7.827.99
(2H, doublet, aromatic protons of PABA), 8.138.24
(1H, singlet, aromatic proton of SA); Number of
5-ASA residues per drug conjugate (UVvisible
spectroscopy, 375 nm, DMSO) equals 3.3.
2.3.2. Synthesis of PAMAMPAHSA conjugates
As described for the synthesis of PAMAM
PABASA conjugate, PAMAMPAHSA conjugate
was prepared by p-amino hippuric acid (PAH,
equivalent to 1:64 ratio of PAMAM dendrimer to
PAH). The PAMAMPAHSA conjugate was
purified by dialysis. The product was obtained as
yellow solid (90.2 mg, 36.2% yield) 1 H NMR
spectrum (D 2 O): d 1.993.42 (484 H, multiplet, CH
protons of PAMAM), 3.753.80 (2H, singlet, CH2
protons of PAH), 6.786.88 (1H, multiplet, aromatic
proton of SA), 7.367.62 (3H, multiplet, aromatic
protons of PAH and SA) 7.637.92 (2H, singlet,
aromatic protons of PAH), 7.958.19 (1H, singlet,
aromatic proton of SA); Number of 5-ASA residues
per drug conjugate (UVvisible spectroscopy, 375
nm, DMSO) equals 9.8.
2.3.3. Synthesis of standard PABASA conjugate
A solution of PABA (10 mmol) in 5 ml water and
a solution of NaNO 2 (10 mmol) in 5 ml water were
mixed. The mixture was stirred in the ice bath
(35 8C) for 15 min and acidified with 3 M HCl. The
solution was stirred for a further 5 min, and then
tested for the presence of excess nitrous acid using
potassium iodide / starch indicator paper. Salicylic
acid (SA, 30 mmol) was dissolved in 30 ml of 0.5 M
NaOH and stirred at 35 8C. The diazonium salt
solution was added to this solution and pH was then
adjusted to 10 using 1 M NaOH. The reddish brown
solution appeared and the solution was stirred further
for 30 min. The mixture was acidified with 3 M HCl
and reddishbrown solid was separated. The solid
was filtered and washed with 50% methanol in water
(2.793 g, 97.56%). IR spectrum (KBr): cm 21 3500
2500 (OHn), 1758 (C=On), 1 H NMR spectrum
(DMSO): d 2.023.44 (484 H, multiplet, CH
protons of PAMAM), 6.886.94 (1H, multiplet,
aromatic proton of SA), 7.547.68 (2H, doublet,
aromatic protons of PABA) 7.667.80 (1H, doublet,
aromatic proton of SA), 7.827.99 (2H, doublet,
aromatic protons of PABA), 8.138.24 (1H, singlet,
aromatic proton of SA) UV absorption maxima 375
nm.
2.3.4. Synthesis of standard PAHSA conjugates
As described for the synthesis of PABASA
conjugate, PAHSA conjugate was prepared by PAH
(10 mmol). The PAHSA conjugate was obtained as
yellow solid (3.390 g, 98.73%) IR spectrum (KBr):
cm 21 3675 (NHn), 35002300 (OHn), 1690
(C=On), 1 H NMR Spectrum (DMSO): d 3.753.80
(2H, singlet, CH2 protons of PAH), 6.786.88 (1H,
multiplet, aromatic proton of SA), 7.367.62 (3H,
multiplet, aromatic protons of PAH and SA) 7.63
7.92 (2H, singlet, aromatic proton of PAH), 7.95
8.19 (1H, singlet, aromatic protons of SA) UV
absorption maxima 375 nm.
2.3.5. Number of mole of SA attached to the
PAMAM dendrimer conjugates
Calibration curves were prepared from standard
PABASA and PAHSA conjugates solutions (210
mg / ml) in DMSO and absorbance at 375 nm was
recorded in order to estimate PABASA and PAH
SA moiety in PAMAMPABASA and PAMAM
PAHSA, respectively.
2.4. 5 -ASA release experiments
2.4.1. Incubation of PAMAM conjugates with the
homogenate of stomach or small intestine of rats
Male Wistar rats (250300 g) were sacrificed and
a midline incision was made. Sections of stomach
and small intestine were collected separately,
homogenized, and the homogenate was diluted with
isotonic acetate buffer (pH 4.5) for stomach and with
isotonic phosphate buffer (pH 6.8) for small intestine
to yield a 10% suspension [19]. To a 0.5-g portion of
each homogenate, 2 ml of dendrimer conjugate
solution in pH 6.8 isotonic phosphate buffer (1.302
mM equivalent to 5-ASA) or sulfasalazine solutions
(1.024 mM equivalent to 5-ASA) were added and the
mixture was incubated for 12 h at 37 8C. At selected
time intervals, samples (300 ml) were taken and 50
 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19
ml of 30% (w / v) trichloroacetic acid were added to
stop the reaction. The quantity of drug release was
then analyzed by HPLC.
2.4.2. Incubation of PAMAM conjugates with the
cecal content of rats
The cecal segment of the intestine contents was
cut open and their content was collected and dis-
persed in nitrogen bubbled PBS, pH 6.8, to yield a
10% suspension. Two ml of dendrimer conjugate
solutions (equivalent to 5-ASA 1.302 mM) or sul-
fasalazine solutions (equivalent to 5-ASA 1.024
mM) were each mixed with 2 ml of cecal contents
(PBS, pH 6.8 as control). The mixtures were incu-
bated at 37 8C under anaerobic condition and sam-
ples (150 ml) were taken at selected time intervals up
to 24 h. Fifty ml of 30% (w / v) trichloroacetic acid
were added to stop the reaction. The quantity of drug
release was then analyzed by HPLC.
2.4.3. Sample preparation for HPLC analysis
One hundred ml of the supernatant were applied
on top of a Bond Elut C-18 extraction cartridge (500
mg, 2.8 ml, Lida, USA), which had been precon-
ditioned with methanol, water and phosphate buffer
(pH 6.8). After sample application, the cartridge was
washed with 1 ml phosphate buffer (pH 6.8) and
eluted with 3 ml of the same solution. The final
solution was then analyzed by HPLC. Percent re-
covery equals 83.76%.
2.4.4. HPLC analysis
The liquid chromatography consisted of an LC-
5000 pump (Jasco PU-980), a gradient unit (Jasco
LG-980-025), a detector set at 240 nm (Jasco UV-
975 (UVVis) and an integrator (Waters, USA). The
column (C-18) was a 30033.9 mm, microbondapak
(Waters, USA) The mobile phase was methanol
phosphate buffer, pH 6.8 (5:95, v / v) and the flow
rate was 1 ml / min.
3. Results and discussion
3.1. Synthesis of dendrimer conjugates
Synthesis of PAMAMPABA (or PAH)SA
conjugate consists of two steps: amide bond forma-
5
tion and synthesis of azo compound. PABA and PAH
were selected as the linkers because of their appro-
priate functionalities. They contain carboxylic groups
for amide bond formation with the amino terminals
of PAMAM dendrimers. In addition, their aromatic
amino groups facilitate diazotization and coupling
with SA to yield the desired azo polymeric drug-
delivery system. In the first step, which is amide
bond formation, CDI was used as a coupling reagent
(Fig. 2). According to our preliminary study, the
optimum molar ratio of starting PAMAMPABA
was found to be 1:64. The next step is the diazotiza-
tion on the aromatic amino group of the PAMAM
PABA (or PAH) conjugate with NaNO 2 under acidic
condition and then coupling with SA under basic
condition to yield PAMAMPABA (or PAH)SA
conjugates. It is established that hydroxyl group of
phenolic compounds is the para-directing group in
coupling with diazonium salts [30], thus we can
predict the site that coupling between PAMAM
PABA diazonium salt and SA occur.
The number of moles of SA attached to the
PAMAM dendrimer conjugates was estimated in-
directly by observation of UV absorption of the azo
aromatic moiety at 375 nm in DMSO. The number
reflects theoretical amount of 5-ASA that will be
released from the macromolecular prodrugs. UV
spectroscopy is specific enough for the SA molecule
that is bound to the conjugate because the azo
aromatic moiety absorbs UV radiation at longer
wavelength than the starting materials. The number
of SA residues and the drug contents in the conju-
gates are shown in Table 1.
It is clearly seen that the utilization of PAH as the
spacer provide the polymer conjugates that carry
approximately three times more 5-ASA than using
PABA as the spacer. This may be due to lower steric
hindrance at COOH group of PAH than that of
PABA molecule. Although the molecule of PAMAM
dendrimer G3 consists of 32 amino terminal groups,
the average ratio of 3.3 and 9.8 mol per conjugate for
PABA and PAH spacers, respectively, were found to
be the maximum ratio that still maintained good
water solubility (1 g / ml). Some researchers have
reported the average optimum number of drug
molecules covalently attached to the PAMAM den-
drimers, for example, the average ratio of doxorubi-
cin to PAMAM G4 with 64 amino terminal groups
6 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19

Fig. 2. Synthesis of dendrimer.

was 2:1 [31] and ibuprofen to PAMAM G4 was 3:1 buffers were carried out in vitro at 37 8C compared
[32]. These indicate that the large number of termi- to those of sulfasalazine (Table 2).
nal groups may not be necessary for carrying more On incubation of conjugates with hydrochloric
drug molecules. In contrast, the drug-carrying acid buffer, pH 1.2, and phosphate buffer, pH 6.8 (no
capacity of the dendrimer depends on other factors biological contents), no 5-ASA or other conjugate
such as the chemical structure of the drug, spacer residues was detected during a 12-h period, which
and the carrier, and also the reaction used. indicated that these macromolecular prodrugs were
chemically stable in the environmental pH of the GI
3.2. Drug release studies tract. The same result was obtained when incubated
the conjugates with the homogenate of the stomach
To investigate the stability and drug release of (Table 2). When both conjugates were incubated
dendrimer conjugates in the GI tract, incubation of with the homogenate of the small intestine and the
PAMAMPABASA and PAMAMPAHSA cecal contents for 12 h, it was clearly seen that
conjugates with different biological content and macromolecular prodrug activation took place mostly
in the rat cecum where the bacterial counts are as
Table 1 high as in the human colon. About 23 and 38% of
The number of SA residue attached to the conjugates and the drug the dose was released as 5-ASA from PAMAM
contents
PABASA and PAMAMPAHSA, respectively,
Spacers No. of SA residues Equivalent wt% whereas relatively small amount of 5-ASA (4.5
in drug conjugate of 5-ASA in the 7.2%) was detected in the homogenate of the small
(mol / mol) conjugates
intestine.
PABA 1:3.3 6.64 In the case of dendrimer conjugate incubation with
PAH 1:9.8 14.58
the homogenate of the small intestine, the significant
 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19 7

Table 2
Drug release (%) from dendrimer conjugates after incubation with different biological contents
Incubation PAMAMPABASA PAMAMPAHSA
medium
5-ASA PABASA 5-ASA PAH-SA
Gastric ND ND ND ND
homogenates
Small intestine 4.561.2 3.860.5 7.261.8 12.563.2
homogenates
Cecal contents 23.563.7 0.260.2 38.265.1 0.460.1
Buffer pH 1.2 ND ND ND ND
Buffer pH 6.8 ND ND ND ND
Data shown are the mean6S.D. (n55).
Percent of drug released after incubation of each solution at 37 8C for 12 h.
ND, not detectable.

amount of PABASA (3.8%) or PAHSA (12.5%)
were also found in the medium (Table 2). The
molecules of dendrimer conjugates consist of two
types of chemical bonds, which can be breakdown
by enzymatic degradation in the GI tract: an amide
bond links between the dendrimer and the spacer,
and an azo bond links between the spacer and SA
molecule. These results pointed out that some of the
amide (peptide) bonds links between the dendrimer
and the spacer were dissociate in the small intestine
region, and a number of smaller prodrugs of 5-ASA
(PABASA and PAHSA) were produced. An N-
acyl amide bond derived from aromatic carboxylic
acid and an amino acid has been reported to be
chemically and biochemically stable in the upper
intestine and breakdown in the large intestine by the
microbial action [33]. However, degradability of
peptide bonds in the small intestine contents was
usually occurred due to a significant amount of
peptidase in the brush border of the small intestine
[21].
It is well known that the release of drug from
prodrugs containing azo bond is triggered by enzyme
azoreductase in the colon (Fig. 3). When PAMAM
PABASA and PAMAMPAHSA conjugates were
incubated with the cecal contents, the amount of
5-ASA released from both conjugates was gradually
increased with time up to 24-h period of incubation.
Although the release rates from PAMAMPABA
SA and PAMAMPAHSA were similar, the conju-
gate with PAH linker showed significant higher
amount of initial drug release than the conjugate with
PABA linker. Consequently, the amount of drug
liberated from PAMAMPAHSA was significantly

Fig. 3. Release of 5-ASA from the conjugates.

8 R. Wiwattanapatapee et al. / Journal of Controlled Release 88 (2003) 19
higher than that of PAMAMPABASA conjugate at
each time point. This indicated that the length and
chemical structure of spacers may affect the initial
attack of azo bond by enzyme. About 45 and 57% of
5-ASA were released from PAMAMPABASA and
PAMAMPAHSA conjugates in 24 h, respectively
(Fig. 4). The cleavage of azo bond in both dendrimer
conjugates was much slower than in sulfasalazine
(80% in 6 h). The observed difference in the rate of
cleavage of low molecular weight prodrug, sul-
fasalazine and dendrimer conjugates may probably
be explained by the different structure of the drug
carriers. The highly branched structure of dendrimer
might have an effect on the decrease of enzyme
cleavability to the azo bond, whereas the azo bond of
the small prodrugs such as sulfasalazine could be
easily attacked. From our previous study in vitro,
cationic PAMAM dendrimers showed high tissue
association which may be useful for prolonging GI
transit time [28]. This could be explained by the
strong interaction of negative cell membrane with
cationic polymers. However, in the case of den-
drimer conjugates, the bioadhesion property may be
different because some amino groups are substituted
by PABASA and PAHSA which contained car-
boxyl groups and also the increase of molecular size.
Further in vitro studies and also in vivo biodistribu-
tion studies are required to understand better the
different physicochemical and biological properties
between the dendrimer and dendrimer conjugates.
Fig. 4. Release profiles of 5-ASA from PAMAMPABASA
conjugate (), PAMAMPAHSA conjugate (d) and sul-
fasalazine (m) during incubation with rat cecal content at 37 8C.
Data shown are the mean6S.D. (n55).
4. Conclusion
The water-soluble PAMAM dendrimer-5-ASA
conjugates containing a designed spacer for colonic
delivery were synthesized. The conjugates showed
colon-specific and prolonged release of 5-ASA. The
results suggested that these dendrimers have po-
tential for use as colon-specific drug carriers. The
structural modifications of different spacers between
the drug and dendrimer are needed in future work to
obtain the optimum release rate of the drug from the
conjugates. Synthesis of other conjugates using
PAMAM dendrimer containing different terminal
groups is ongoing.
Acknowledgements
This research was supported by National Science
and Technology Development Agency, Thailand. We
thank Professor Ruth Duncan for her valuable ad-
vice, Mr. Pisit Buaking for helping with animal
works and Dr. Sanae Kaewnopparat for providing
some useful equipments.
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