Novel salicylazo polymers for colon drug delivery: Dissolving polymers by means of bacterial degradation

Article abstract of DOI:10.1002/jps.21875

Novel azo polymers were prepared for colonic drug delivery with a release mechanism based on structural features of azo derivatives designed for rapid bacterial degradation leading to soluble polymers. Two Salicylazo derivatives were prepared and conjugated as side chains at different ratios to methacrylic acid-methyl methacrylate copolymers (Eudragits). The azo compounds were designed to have a hydrophilic and a hydrophobic part on opposite sides of the azo bond. Upon reduction of the azo bonds, the hydrophobic part is released, resulting in a more water soluble polymer. The solubility of the polymeric films was studied relative to Eudragit S known to dissolve toward the end of the small intestine. One of the two azo derivatives prepared gave rise to polymers, which showed reduced solubility relative to Eudragit S. These polymers were subjected to reduction tests in anaerobic rat cecal suspensions by following the release of the hydrophobic product. Reduction rate was found to be rapid, comparable to that of Sulfasalazine. Studies on the azopolymeric films in anaerobic rat cecal suspensions, showed that these polymers dissolve faster than in sterilized suspensions. Solid dosage forms may be coated with these polymers to provide an efficient delivery system to the colon with a rapid release mechanism.

Full text of DOI:10.1002/jps.21875

Novel Salicylazo Polymers for Colon Drug Delivery:
Dissolving Polymers by Means of Bacterial Degradation
SIGAL SAPHIER, YISHAI KARTON
Department of Organic Chemistry, Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 74100, Israel
Received 12 January 2009; revised 25 May 2009; accepted 7 June 2009
Published online 14 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21875
ABSTRACT: Novel azo polymers were prepared for colonic drug delivery with a release
mechanism based on structural features of azo derivatives designed for rapid bacterial
degradation leading to soluble polymers. Two Salicylazo derivatives were prepared and
conjugated as side chains at different ratios to methacrylic acid—methyl methacrylate
copolymers (Eudragits). The azo compounds were designed to have a hydrophilic and a
hydrophobic part on opposite sides of the azo bond. Upon reduction of the azo bonds, the
hydrophobic part is released, resulting in a more water soluble polymer. The solubility of
the polymeric films was studied relative to Eudragit S known to dissolve toward the end
of the small intestine. One of the two azo derivatives prepared gave rise to polymers,
which showed reduced solubility relative to Eudragit S. These polymers were subjected
to reduction tests in anaerobic rat cecal suspensions by following the release of the
hydrophobic product. Reduction rate was found to be rapid, comparable to that of
Sulfasalazine. Studies on the azopolymeric films in anaerobic rat cecal suspensions,
showed that these polymers dissolve faster than in sterilized suspensions. Solid dosage
forms may be coated with these polymers to provide an efficient delivery system to the
colon with a rapid release mechanism. ß 2009 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci 99:804–815, 2009
Keywords: biodegradable polymers; colon; colonic drug delivery; controlled delivery;
oral drug delivery; polymeric drug delivery systems; reduction
INTRODUCTION
reactions carried out by the microbiota residing in
the colon and has the potential of being the most
specific.9 Mainly two types of reactions have been
used for the construction of many colon specific
delivery vehicles; hydrolysis of polysacchrides10
and reduction of azo bonds.11 These reactions
have been used for the development of prodrugs,
hydrogel matrices, and polymeric coatings.
Using azo bonds for polymeric coating, one may
design a variety of azo compounds with different
molecular structures and combine them with a
choice of polymers possessing appropriate film
forming properties. Azo bonds have been incorpo-
rated in the backbone or as crosslinks using
several different synthetic polymers in efforts to
develop coatings of dosage forms for colonic
delivery. Polymers in which azo bonds were
incorporated as crosslinkers include copolymers
of styrene and 2-hydroxyethyl methacrylate12,13
Delivery of drugs to the colon via the oral route
has many important therapeutic applications.
These include local disease of the colon such as
Crohn’s disease, ulcerative colitis, irritable bowel
syndrome, and colon cancer.1 In addition, the
colon has been suggested as a site for systemic
delivery of proteins, peptides and other labile
molecules2,3 and for chronopharmaceutics.4 Deliv-
ery to the colon may be achieved by several
different approaches such as time,5 pH,6 pres-
sure,7 and biodegradation8 based systems. Biode-
gradation is achieved by exploiting the unique
Correspondence to: Sigal Saphier (Telephone: 972-8-
9381740; Fax: 972-8-9381548; E-mail: sigals@iibr.gov.il)
Journal of Pharmaceutical Sciences, Vol. 99, 804–815 (2009)
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
804
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NOVEL SALICYLAZO POLYMERS FOR COLON DRUG DELIVERY
805
and copolymers of 2-hydroxyethyl methacrylate
and methyl methacrylate.14,15 Other polymers
were prepared incorporating azo bonds into the
main chain of the polymer including poly(ether
ester) azo polymers,16 polyurethanes,17,18 and
polyamides.19,20
These azo polymers were designed with little
regard to the structural features of the azo
compound, features that could accelerate the rate
of reduction. Bifunctional azo aromatic com-
pounds were synthesized with the functional
groups used only for copolymerization. No addi-
tional functionality was added to the aromatic
rings. Slow or incomplete reduction (stopping at
the hydrazo stage) of these simple structures
could be part of the reasons for the seemingly slow
microbial degradation and drug release found in
some of these polymers.11
The rational for drug release in the above
mentioned polymeric systems was that reduction
of the azo linkage in the polymer backbone or
crosslinks forms pores in the polymeric film
enabling diffusion of the drug or alternatively
causing collapse of the polymeric structure lead-
ing to erosion or rupture of the coating and release
of the drug load.
Figure 1. Design of azo polymers that become more
soluble upon reduction by colonic bacteria.
Azo compounds attached as side chains (instead
of crosslinks or backbone) have been used before
in the development of hydrogels.21 The proposed
mechanism of release from these hydrogels was
similar to the mechanisms of drug release
described above and was based on cleavage of
the azo bond causing loosening of the polymeric
matrix leading to the release of drug.
polymer, leaving a more soluble polymer, which
can dissolve at the pH of the colon.
In addition to the new release mechanism
designed, and in order to obtain an azo polymer
that will be degraded at a fast and physiologically
relevant rate by colonic bacteria, the azo com-
pounds were designed specifically for fast degra-
dation. Various papers have been published on the
structure-activity relationship of azo reduction by
bacterial species.22–27 Yet, since this processes
involves many different bacteria in vivo, it is
difficult to make definite predictions about the
rate of reduction of a specific azo compound.28
Therefore, any azo compound designed should be
tested for rate of reduction compared to a known
model prior to its use in the preparation of a new
polymer. In this work two azo compounds were
designed and tested for degradation using
rat cecal contents29 prior to their use for the
preparation of novel polymers.
In this work we suggest a new means for drug
release based on rapid bacterial azo degradation
leading to pH dependent soluble polymers. We
have designed azo compounds to have one
hydrophilic aromatic ring and one hydrophobic
ring. Then, they were conjugated as side chains to
copolymers of methacrylic acid and methylmetha-
crylate 1:1 (Eudragit L) and 1:2 (Eudragit S)
(Fig. 1). Eudragit L and S are used as enteric
coating agents since they are resistant to gastric
fluid. Eudragit L is soluble at pH > 6 (correspond-
ing to the pH at the jejunum) and Eudragit S is
soluble at pH > 7 (corresponding to the pH at the
ileum). The azo-Eudragit conjugates were
designed to make them less soluble than Eudragit
S so they will survive the entire pH range of
the gastrointestinal tract including the slightly
alkaline environment of the ileum. Reduction of
the azo linkage by bacteria in the colon releases
the hydrophobic part of the azo molecule from the
MATERIALS AND METHODS
Materials and Measurements
Eudragit S-100 (ES) and Eudragit L-100 (EL)
¨
were obtained as a gift from Evonik ROHM
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SAPHIER AND KARTON
GmbH, (Darmstadt, Germany). Potassium iodine/
starch paper was purchased from Macgerey-Nagel
GmbH&Co. KG. (Du¨ren, Germany), Sulfapyri-
dine from Fisher Scientific (Pittsburgh, PA),
solvents and salts from Merck (Darmstadt,
Germany) and all other chemicals and reagents
from Sigma–Aldrich (Rehovot, Israel).
evaporated and the mixture was dissolved in
water. The solution was acidified with concen-
trated hydrochloric acid until precipitation of
product was observed. The mixture was cooled
to 48C for further precipitation of product.
The product was filtered and the process of
dissolution and solidification was repeated several
times until NMR showed no traces of ethylene
diamine. 1H NMR (NaOD in D2O, 300 MHz):
8.17 (d, J ¼ 2.8 Hz, 1H), 7.50 (dd, J1 ¼ 9.1 Hz,
J2 ¼ 2.8 Hz, 1H), 7.42 (d, J ¼ 8.45 Hz, 2H), 7.27 (m,
3H), 6.54 (d, J ¼ 9.1 Hz, 1H). UV-Vis (PB
buffer pH 7.4) lmax ¼ 385 nm.
1H NMR spectra were recorded on an Avance
Bruker 300 (at 300 MHz) and/or Avance Bruker
500 (at 500 MHz) spectrometer. UV-Vis spectra
were measured using an Amersham Pharmacia
Biotech (Cambridge, UK) Model Ultraspec 2100
Pro.
Synthesis
3-(2-Amino-ethylcarbamoyl)-4-Hydroxy-5-
Phenylazo-Benzenesulfonic Acid (6)
2-Hydroxy-5-Phenylazo-Benzoic Acid (1)
A similar procedure was followed. The product
containing Ethylene diamine residue was dis-
solved again in methanol. Di-tert-butyl dicarbo-
nate (2 g, 9 mmol) was added to the methanolic
solution to eliminate residues of ethylene diamine.
The mixture was left over night in refrigeration.
The solvent was then evaporated and the solid
was dissolved in water (30 mL) and the excess of
protected ethylene diamine was extracted with
hexane (40 mL Â 2) and Ethyl acetate (70 mL Â 3).
The water phase was evaporated in vacuum and
the crude product was dissolved again in water
and acidified with concentrated hydrochloric acid.
After precipitation of the product, it was filtered
and dried to give 215 mg solid product.1H NMR
(NaOD in D2O, 300 MHz): 8.27 (d, J ¼ 2.7 Hz, 1H),
7.80 (dd, J1 ¼ 8.4 Hz, J2 ¼ 1.5 Hz, 2H), 7.77 (d,
J ¼ 2.7 Hz, 1H), 7.46 (m, 3H), 3.42 (t, J ¼ 6.2 Hz,
2H), 2.75 (t, J ¼ 6.2 Hz, 2H). UV-Vis (PB buffer pH
7.4) lmax ¼ 226 nm.
Standard procedure of azo synthesis by diazonium
coupling was followed.30 The product precipitated
in the form of an orange powder. 1H NMR (NaOD
in D2O, 300 MHz): 7.67 (d, J ¼ 2.8 Hz, 1H), 7.57
(m, 3H), 7.42 (m, 2H), 7.34 (t, J ¼ 7.3 Hz, 1H), 6.53
(d, J ¼ 9.0 Hz, 1H). UV-Vis (PB buffer pH 7.4)
lmax ¼ 230, 347 nm.
2-Hydroxy-3-Phenylazo-5-Sulfo-Benzoic Acid (2)
The product was obtained in the form of a brown-
red solid. 1H NMR (D2O, 500 MHz): 8.26 (d,
J ¼ 2.1 Hz, 1H), 8.10 (d, J ¼ 2.1 Hz, 1H), 7.75 (m,
2H), 7.47 (m, 3H). UV-Vis (PB buffer pH 7.4)
lmax ¼ 230, 309 nm.
2-Hydroxy-5-Phenylazo-Benzoic
Acid Methyl Ester (3)
A solution of 1 (2.25 g) and BF3ÁO(C2H5)2 (2 mL) in
methanol was refluxed for 16 h. Methanol was
evaporated under vacuum and ice, water and
sodium hydroxide were added. The reaction
mixture was acidified to pH 7 and the product
precipitated to give 4 g of a wet solid. The product
was taken to the next step without further
characterization.
Conjugation of Azo Compounds to
Eudragit Polymers
A typical procedure is as follows: Polymer EL-7-1:
Azo compound 5 (100 mg, 0.35 mmol) was dissolv-
ed in dry DMF (2 mL). Eudragit L-100 (450 mg)
was dissolved in DMF (4 mL) and was added to the
solution of 5. o-(benzotriazol-1-yl)-N,N,N0,N0-tet-
ramethyluronium hexafluorophosphate (HBTU)
(132 mg, 0.35 mmol) in DMF (2 mL) and triethy-
lamine (0.15 mL, 0.12 mmol) were then added to
the mixture. The reaction was stirred at room
temperature for 12 h. The DMF was evaporated
and the mixture was dissolved in alkaline aqueous
solution, which was then acidified and centrifuged
to precipitate the resulting polymer. This process
2-Hydroxy-3-Phenylazo-5-Sulfo-Benzoic
Acid Methyl Ester (4)
A similar procedure to 3 was followed.
N-(2-Amino-Ethyl)-2-Hydroxy-5-Phenylazo-
Benzamide (5)
The ester 3 was partly dissolved in Methanol
(70 mL). Excess of Ethylene diamine (15 g) was
added to the solution. The reaction mixture was
heated to 608C for 4 h. The solvent was partly
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807
was repeated three times. The conjugation ratio
was determined for each polymer by 1H NMR
signal integration. Polymer EL-7-1 obtained was
Reduction Tests of Soluble Molecules in Anaerobic
Cecal Suspension
Two millimolar solutions of the small molecules 1,
2, 2-amino 5-phenylazo benzene sulfonic acid
(APAS), Acid red-1, Methyl Orange, Amaranth
and Sulfasalazine and the polymers EL-7-1 and
EL-8-1 were prepared in phosphate buffer
100 mM, pH 7.4. In some cases dissolution was
aided by stirring and sonication. In cases where it
was not possible to obtain a concentration of 2 mM
due to insolubility of the compounds, solutions of
1 mM were prepared and the volume of cecal
suspension was adjusted to obtain an identical
final concentration of the azo compound. Soluble
polymers were dissolved to obtain a maximal
concentration possible of 1 mM. For each com-
pound duplicates were prepared by adding 0.4 mL
of stock solution into 4 mL amber color vials. At
time zero 3.6 mL of cecal suspension and 40 mL of
20 mg/mL benzyl viologen (BV) were added (some
experiments were also performed without BV),
the vials were sealed by rubber septum and the
mixture was bubbled with nitrogen for 2 min. The
vials were incubated anaerobically at 378C with
shaking. The final concentrations were 90 mg/mL
rat cecal contents, 2.25 mg/mL a-D-glucose,
0.2 mg/mL BV and 0.2 mM azo-compound. Reduc-
tion products such as aniline and sulfapyridine
were also subjected to the same conditions to test
their stability in cecal contents.
1
characterized by H NMR to have the following
ratio of substituents etser/acid/azo 50:45:5. 1H NMR
(D2O þ NaOD, 300 MHz):6.6–8.3 (m, ArH from azo
conjugate), 3.5 (broad s, CH3 of ester with CH2 of
ethylenic linker), 2.8 (s, CH2 of ethylene linker)
0.5–1.8 (broad m, CH2 and CH3 of polymer
backbone). UV-Vis (PB buffer pH 7.4) lmax
345 nm.
¼
Chemical Reduction of Azopolymer
Polymer EL-7-1 (70 mg) and sodium bicarbonate
(74 mg) were dissolved in water (2.1 mL). Sodium
chloride (70 mg) and sodium dithionite (Na2S2O4)
(135 mg) were also dissolved in water (3.5 mL).
The reducing solution was added to the polymer
solution and the mixture was sonicated for 30 min.
The reaction mixture was left at room tempera-
ture over-night. The reaction mixture was then
acidified and the polymer was separated by
centrifugation. Acidification and centrifugation
was repeated several times to obtain a clean
product EL-9. 1H NMR (NaOD, 500 MHz): 6.5–7.2
(ArH of remaining ring), 3.5 (broad s, CH3 of ester
with CH2 of ethylenic linker), 0.6–1.8 (CH2
and CH3 of polymer backbone).
Preparation of Anaerobic Cultures from
Rat Cecal Contents
Sample Preparation and HPLC Analysis
The institutional ethics committee approved the
protocol.
A
solution of phosphate buffer
At predetermined time intervals ranging from
30 min to 5 h, samples (0.7 mL) were withdrawn
from the incubation mixtures using a 18G syringe
under nitrogen bubbling. The samples were
acidified with 0.15 mL of 1 M hydrochloric acid
to obtain a pH of about 3. The samples were
centrifuged for 5 min at 10,000 rpm and filtered
through a FVDF Whatman 13 mm syringe filter
GD/X with a glass prefilter. Samples were either
taken immediately to HPLC analysis or frozen at
ꢀ208C until analysis.
(100 mM, pH 7.4)26,31,32 was prepared and deox-
ygenated by bubbling nitrogen for 10 min. Male
Sprague–Dawley rats were sacrificed and the
cecum was exposed. The cecum contents were
removed and used to make a 10% w/v suspension
in the oxygen free buffer. A solution of 250 mg/mL
a-D-glucose in PB was also prepared and deoxy-
genated. Glucose (1% v/v) solution was added to
the suspension and nitrogen was bubbled for
additional 10 min. The bottle was tightly sealed to
provide anaerobic conditions and the suspension
was incubated at 378C over-night33 with shaking
using a shaker-incubator from MRC model TU-
400. For controls, cecal contents were sterilized13
in a pressure tight vessel by heating to 1208C for
20 min using a laboratory microwave. The ster-
ilization was tested by following the reduction of
sulfasalzaine in the strile cecal contents. No
reduction was observed in sterile cecal contents.
HPLC was performed on a Waters HPLC
system fitted with a Waters 2487 Dual wavelength
absorbance detector and Waters 717 plus auto-
sampler. A gradient method was developed to
allow the different compounds to be analyzed by
the same method. The solvents were 40 mM
potassium phosphate buffer pH ¼ 7.4 þ 0.1%
tetrabutylammoniumhydroxide and acetonitrile
from 83% to 70% buffer. A C18 RP column (Gemini
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110A, 150 Â 4.6 mm, 5 m mean particle size,
Phenomenex) was used. For each compound
tested, the chromatogram was followed at two
wavelengths corresponding to lmax of the starting
azo compound and aniline product as determined
by measuring the compounds UV-Vis spectra.
Product concentration was determined by
external standard. Linearity of product concen-
tration under the same conditions as the experi-
ment was shown for aniline (R2 ¼ 0.9996) and
sulfapyridine (R2 ¼ 0.9938). For each azo com-
pound, only one of the two products was followed
depending on commercial availability and method
developed. The reduction of 1, 2, EL-7-1, EL-8-1,
Acid red-1 and 2-amino-5-phenylazo benzene
sulfonic acid was evaluated by following the
cleavage product aniline. Sulfapyridine was used
to follow sulfasalazine, 4-Amino-1-naphthalene
for amaranth and dimethylamino aniline for
methyl orange.
ometer and absorption at lmax was measured
using quartz quvettes. Samples were then
returned to the vial. The experiment continued
until a maximal absorption was observed and the
both films dissolved completely.
Solubility of Azopolymer Versus Eudragit S
Experiments involving Eudragits, which do not
have meaningful UV-Vis absorptions, were fol-
lowed by measuring film thickness. The film
thickness was measured using a Mitutoyo digital
micrometer model MDC-25S with a 0–25 mL
range and an accuracy of 0.001 mL. Film thick-
ness varied between experiments according to the
volume and concentration of the solution used.
Duplicate slides were taken out of solution at
three time points predetermined by preliminary
experiments, washed with deionized water, dried
over-night in a hood and their thickness was
measured. Percent of polymer dissolved was
calculated for each slide as the ratio between
the thickness of the film before and after the
experiment.
Casting of Films
Azopolymers and Eudragits were dissolved in
DMF to give a 10% or 20% w/w solution. Glass
slides were cut using a diamond knife to fit the
vials used during the experiments described in the
following sections. Depending on the experiment,
6–8 mL drops were cast using micropipette on
glass slides which were either transparent or
frosted. The glass slides were left to dry over-night
in a hood and circular films with a diameter of
about 0.5–1.0 cm were formed. Dryness of films
was confirmed by obtaining constant weight.
Degradation Test of Polymeric Films by
Rat Cecal Contents
Each glass slide was inserted back to back with
another slide into a 20 mL scintillation vial. A
suspension of anaerobic culture of cecal contents
(7 mL) adjusted to pH 6.8 and BV (20 mg/mL,
0.07 mL) were added and the vial was sealed with
septum and bubbled with nitrogen for 2 min.
Controls were prepared in the same way, replac-
ing cecal contents with sterilized cecal contents
and the vials were closed with regular caps. Vials
were then incubated at 378C with shaking
(100 rpm). At predetermined time points, tripli-
cates were opened, the films were washed gently
with deionized water and left to dry in a hood
overnight. The thickness of the remaining film
was measured the next day. Percent of polymeric
film dissolved was calculated for each slide as the
ratio between the thickness of the film before and
after the experiment.
Solubility Tests of Polymeric Films
All solubility tests were performed at 378C with
shaking.13 The shaking rate was adjusted accord-
ing to the type of test and vessels used to provide
efficient stirring (between 100 and 200 rpm,
usually 150 rpm). Preliminary tests were pre-
formed by visually examining the films which
were cast on small glass slides in Petri dishes
containing buffers at different pH’s.
Solubility of Chemically Reduced Film Versus
Azopolymer
RESULTS AND DISCUSSION
Preparation of Azo Derivatives of Salicylic Acid
Experiments were performed in vials filled with
15 mL buffer. Samples (0.5 mL) were taken at
predetermined time intervals to the spectrophot-
Two aromatic azo compounds were designed
(Fig. 2) and prepared. For each compound, one
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duced by the aromatic rings and loss of free
carboxylic acid.
Compounds 1 and 2 were synthesized following
standard procedures.30 Aniline was diazotized
and coupled with salicylic acid and 5-sulfosalicylic
acid separately, under slightly basic conditions
(Fig. 3). For the conjugation of compounds 1 and 2
to polymers, they were esterified to give 3 and 4,
which were directly reacted with excess ethyle-
nediamine in methanol to obtain amido-amines 5
and 6. Care was taken to remove all ethylene
diamine before conjugation of 5 and 6 to the
Eudragit copolymers to avoid cross-linking. This
was done by dissolving the product in water,
acidifying the solution with concentrated hydro-
chloric acid and filtration of the product. The
process was repeated several times until 1H NMR
spectra showed no remaining ethylene diamine.
In the case of azo compound 6, it was necessary to
react the product with Di-tert-butyl dicarbonate
and consequently remove Boc protected ethyle-
diamine. The protecting group on the product was
then hydrolyzed back to the free amine.
Figure 2. Azo compounds based on amino salicylic
acid.
unsubstituted aromatic ring was introduced,
acting as a model for a hydrophobic moiety. This
model compound may be modified in the future
with other anilinic derivatives having more
favorable pharmacological properties. The second
aromatic ring was substituted with hydrophilic
groups. Compound 1 is a combination of aniline
and 5-aminosalycilic acid, which is the active
ingredient in Sulfasalazine. Sulfasalazine is an
azo prodrug, which has been in clinical use for
more than 50 years and its reduction is known to
be fast and efficient.
Compound 2 was designed with the addition of a
sulfonic acid to the hydrophilic ring in order to
serve two purposes. First, sulfonic acid, being an
electron-withdrawing group should assist in
obtaining faster degradation rates by lowering
the electron density at the azo bond.23 This was
shown for example by Kopecek and coworkers34
who have designed hydrogels and polymeric
prodrugs having a halogenic constituent on the
azo-aromatic ring to accelerate the rate of azo
reduction. In addition, a certain level of hydro-
philicity is necessary for the azo-reductases to act
upon the azo groups of the polymer.24 Sulfonic
acid would increase the water solubility of the
polymer, counteracting the hydrophobicity intro-
Reduction of Azo-Salycilic Derivatives by
Rat Cecal Contents
Azo compounds 1 and 2 were subjected to reduction
tests in suspensions of rat cecal contents. Rapid
reduction was a prerequisite for the use of
these compounds for conjugation to Eudragit
copolymers. Sulfasalazine was chosen as the
reference compound. Two other azo dyes which
are common reference materials; Amaranth13,17
and Methyl orange31,35 were also used. In addition,
Acid red-1 and 2-amino-5-phenylazo benzene
Figure 3. Synthesis of azo-salicylic acid derivatives.
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sulfonic acid (APAS) were also examined since they
have one side of the azo bond bearing an
unsubstituted aromatic ring, similar to compounds
1 and 2. The disappearance rate in HPLC of the
parent azo compounds was usually faster than the
appearance of the anilinic products. This demon-
strates that reduction to the hydrazo form is faster
than the second step of reduction and cleavage of
the hydrazo bond to anilines. Rate of reduction
should not be measured by UV spectroscopy which
measures the disappearance of the color formed by
the azo conjugated bond. Such measurements do
not confirm degradation to the corresponding
anilines since the reduction can stop at the hydrazo
stage.18
Surprisingly the rate of reduction of 1 in
anaerobic rat cecal suspension exceeded that of
Sulfasalazine (Fig. 4). Compound 2 was reduced at
a rate slightly lower than Sulfasalazine. Both
compounds were reduced at a rate faster than
the common reference compounds Amaranth and
Methyl orange. The two additional compounds
containing an unsubstituted aromatic ring
showed very low or no reduction. The above
finding demonstrates the importance of examin-
ing the model azo compounds before preparation
of azo polymers since not all small azo molecules
are reduced at a similar and sufficient rate.
Following the successful results showing rapid
reduction rates of compounds 1 and 2, a linker was
attached to the molecules (Fig. 3) and they were
conjugated to Eudragit copolymers as described in
the next section.
Conjugation to Copolymers and
Chemical Reduction
The salicylazo compounds were conjugated at
different ratios to Eudragit L and Eudragit S to
give a total of six different polymers (Tab. 1).
Compound 5 was conjugated to Eudragit L at
three different ratios (EL-7-1 to EL 7-3) and to
Eudragit S (ES-7-4) in one ratio. Compound 6 was
conjugated only to Eudragit L (EL-8-1 and EL-8-
2) at two different ratios.
Conjugation of 5 and 6 to the free carboxylic
acids of Eudragit L and S was performed using
HBTU as the condensation reagent, in the
presence of triethylamine in DMF (Fig. 5). The
degree of azo conjugation was determined by
the integration ratio between aromatic versus
1
backbone hydrogens by H-NMR.
Initially polymers EL-7-1 and EL-8-1 were
prepared and their solubility and reduction rate
was examined as will be described in the following
sections. Higher substitution polymers EL-7-2,
EL-7-3, and EL-8-2 were prepared in order to
further reduce the solubility of the azo polymers.
Eudragit S was used for the preparation of an even
less soluble polymer, the azo conjugate ES-7-4
(Tab. 1).
Theoretically the reduction of the prepared azo
polymers should lead to more soluble polymers. In
order to simulate the properties of the azo polymer
after bacterial reduction, prior to engaging in
ex vivo experiments, chemical reduction of EL-7-1
and EL-8-1 was performed to obtain polymers
EL-9 and EL-10 (Fig. 5) using sodium dithionite
according to a known procedure.36
Reduction of Dissolved Polymers by Rat
Cecal Contents
Due to the relatively low rate of azo conjugation,
polymers EL-7-1 and EL-8-1 were successfully
dissolved in phosphate buffer pH 7.4. This enabled
us to perform a reduction test similar to the one
performed with small molecules and thus compare
their rates of reduction. The azo reduction rate in
cecal contents was tested and again, Sulfasalazine
was used as the reference compound. For both
polymers, the rate of aniline formation was faster
than the rate of sulfapyridine formation from
sulfasalazine (Fig. 6). This result was obtained
only in the presence of the electron carrier benzyl
viologen (BV). In the absence of the electron
carrier, polymers were degraded at a much
slower rate. It has been shown by Kopecek and
Figure 4. Percent reduction of different azo com-
pounds after 50 min in cecal contents. The experiment
was carried out without the addition of BV and was
performed in duplicates.
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NOVEL SALICYLAZO POLYMERS FOR COLON DRUG DELIVERY
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Table 1. Azo Polymers Prepared by Conjugation of Azo Compounds 5 and 6 to
Eudragit Copolymers
Polymer Number Azo Derivative Unit Ratio Obtained m/n/k Copolymer Type
EL-7-1
EL-7-2
EL-7-3
ES-7-4
EL-8-1
EL-8-2
5
5
5
5
6
6
50:45:5
50:40:10
50:36:14
67:27:6
50:45:5
50:42:8
Eudragit L
Eudragit L
Eudragit L
Eudragit S
Eudragit L
Eudragit L
coworkers35 for hydrogels crosslinked by aromatic
azo compounds that the rate of reduction in vitro
after addition of BV is comparable to the rate of
reduction of implanted gel disks in vivo. Thus,
addition of an electron carrier is significant for
obtaining an in vitro system in which the rate of
azopolymer reduction is physiologically relevant.
Under these conditions both polymers showed a
remarkably rapid reduction rate, all azo groups
being reduced within a few hours.
Polymer EL-8-1 bearing a sulfonic group was
reduced at a higher rate than EL-7-1. This result
seems to contradict the results seen with the free
azo compounds where 1 was reduced at a faster
rate than 2. The experiment was repeated,
showing similar results. It is possible that within
the polymer, the added solubility provided by the
sulfonic acid is of importance for accessibility of
the reducing agents to the azo bond.
Effect of Azoconjugation on Solubility
of the Polymers
Once it was shown that these azopolymers could
undergo rapid reduction by the bacteria in rat
cecal contents, we turned to examine their
physical properties. In order to implement our
concept, the polymer has to remain intact until
arrival at the colon. Eudragit S provides protec-
tion throughout the stomach and most of the small
intestine. It dissolves toward the end of the ileum
were the pH rises above 7. Therefore, in order to
survive the ileum and arrive at the colon intact,
the azopolymers designed should have solubility
slightly less than Eudragit S. On the other hand
the solubility should not be too low to allow
accessibility of polymeric side chains to the
reducing agents. Preliminary tests were per-
formed on all polymers by visual examination of
Figure 5. Synthesis of azo polymers and chemical reduction.
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Figure 6. Reduction of soluble polymers EL-7-1 and
EL-8-1 in rat cecal contents with addition of BV. The
reduction was evaluated by following aniline formation
using HPLC and was compared to sulfasalazine by
following sulfapyridine formation. The slight seeming
decrease in percentile of aniline after arriving at 100%
reduction is within the error range of the experiment
resulting from the nonhomogenous character of the
cecal contents suspension. The experiment was per-
formed in duplicates. (~) EL-8-1, (*) EL-7-1, and
(&) Sulfasalazine.
Figure 7. Percent of dissolved polymer EL-7-2 com-
pared to Eudragit S at pH 7. The experiment was
performed in duplicates. Film thickness was
40 ꢁ 5 mm. (&) EL-7-2 and (&) Eudragit S100.
lack of uniformity on the surface of the film, the
differences in polymers can clearly be seen.
The measurements were used to evaluate the
solubility of the polymeric films qualitatively by
comparing between the new polymers to the
known Eudragit S.
polymeric films at pH 1 representing the stomach
(0.1 N HCl) and at several pH values at the range
of 6.6–7.5. All polymers showed resistance to the
acidic pH for at least 2 h, similar to Eutragits.
Polymers EL-8-1 and EL-8-2 (containing sulfonic
acid) showed similar solubility to that of Eudragit
L. The higher ratio of azo compound in polymer
EL-8-2 relative to EL-8-1 did not seem to decrease
it is solubility. Apparently the sulfonic acid more
than compensates for the addition of the aromatic
ring and conjugation through the carboxylic
group. Thus, this azo compound gives rise to
polymers that are too soluble for our purpose and
could not be used as is for delivery to the colon
despite the excellent bacterial reduction rate of
EL-8-1. The following experiments were therefore
focused on the copolymers conjugated to the azo
derivative of salicylic acid (EL-7).
The solubility of EL-7-2 was slightly lower than
Eudragit S. Polymer EL-7-3 was prepared with a
higher ratio of azo compound conjugated to
Eudragit L and solubility was examined in a
separate experiment (Fig. 8). The solubility of EL-
7-3 was much lower than Eudragit S, as expected
from the higher rate of azo derivative on the
polymer compared to EL-7-2. Both EL-7-2 and
EL-7-3 dissolved at a rate slower than Eudragit S.
Next, we wanted to examine the solubility of an
azo polymer based on conjugation of 5 to the less
soluble Eudragit S. Films of polymer ES-7-4 did
not dissolve at all under the pH range used for
Polymers EL-7-1 to EL-7-3 showed a promising
trend of solubility in preliminary tests. The higher
the ratio of azo to carboxylic acids, the lower the
solubility became. EL-7-1 showed solubility simi-
lar to Eudragit S while EL-7-2 and EL-7-3 showed
decreasing solubilities.
The solubility of polymeric films made of EL-7-2
was compared to that of Eudragit S (Fig. 7). Since
Eudragit S does not have any meaningful UV-Vis
absorption, this was evaluated by measuring the
thickness of polymeric films cast on frosted glass
slides at several time points. Although this
measurement may not be very accurate due to
Figure 8. Percent of dissolved polymer EL-7-3 at pH
7 and pH 7.4. The experiment was performed in dupli-
cates. Film thickness was 60 ꢁ 5 mm. (&) EL-7-3 (pH 7),
(&) Eudragit S (pH 7), (^) EL-7-3 (pH 7.4), and (^)
Eudragit S (pH 7.4).
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NOVEL SALICYLAZO POLYMERS FOR COLON DRUG DELIVERY
813
polymers EL-7-2 and EL-7-3 and up to pH 7.8 for
at least 8 h. It was further evaluated at pH 8.2
(partially dissolved) and at pH 8.6 (completely
dissolved). Such high pH requirement for dissolv-
ing the polymer seems to make this particular
polymer too insoluble for our purpose, an estima-
tion that proved correct once degradation in cecal
contents was examined.
From the six polymers prepared and evaluated,
polymers EL-7-2 and EL-7-3 had the most
promising properties. On one hand, they showed
less solubility than Eudragit S, which is necessary
in order to survive the ileum intact. On the other
hand, they were not completely insoluble in
this pH range, which is important for being
accessible to reduction. Future optimization of
film thickness and ratio of these two polymers will
lead to a coated capsule which will remain intact
until arrival at the colon.
Figure 9. Percent of dissolved polymeric film of EL-
7-1 compared to its reduction product EL-9 in phos-
phate buffer pH 6.6. The process was followed by wave-
length absorption of the solution. The absorbance of the
reduced polymer, after loosing the azo chromophore was
lower then the azopolymer with a maximal OD of
around 0.04. This lead to a larger error range of the
points measured compared to the parent azo polymer,
which is reflected by the fluctuations seen after the
reduced polymers were completely dissolved at
30 min. The experiment was performed in duplicates.
Film thickness was 20 ꢁ 5 mm. (*) EL-7-1 and (*) EL-9
(reduced).
Effect of Chemical Reduction on Solubility
of an Azopolymer
Subsequent to arrival of an intact polymer to the
colon and its reduction, a prerequisite to success-
ful drug release would be that the reduced form of
the polymer would be more soluble than the
unreduced polymer. Therefore, in order to confirm
this concept, the solubility of EL-9, a film of the
chemically reduced polymer EL-7-1 was com-
pared to that of the unreduced parent azo
polymer. Since both polymers have UV absorp-
tion, the process was followed by UV spectroscopy
of the solution (Fig. 9). It can be seen from the
figure that films of the reduced polymer EL-9
dissolved much faster than films of the parent
polymer EL-7-1. The reduced film EL-9 was
practically dissolved after 30 min while it took
almost a 100 min for the parent film to completely
dissolve under the same conditions.
slides and the solubility of these polymers was
tested in rat cecal contents with the pH adjusted
to 6.8. Sterilized cecal contents at the same pH
were used as control. The use of sterile cecal
contents as a control instead of a buffer or a
culture growth media insured that the difference
in solubility observed is only due to reduction of
the azo bonds by reductase activity and not due to
physical abrasion from solid materials or other
non bacterial effects of the luminal contents.13 In
both polymers it can be seen that the rate in which
the polymeric films dissolved in cecal contents is
significantly higher than in cecal contents that
have been sterilized (Fig. 10). These results are
due to the activity of the azoreductases, which
reduce the azo bonds leaving a more soluble
polymer. In the previous section it was shown that
these films are less soluble than Eudragit S and
would thus survive the conditions of the ileum. In
conditions that simulate the colon (rat cecal
suspensions) these polymers are shown to be
degraded by reduction and subsequently dissolve
significantly more rapidly than in the absence of
reducing agents. The combination of these results
shows that the polymers may be used for the
delivery of solid dosage forms to the colon with
rapid release.
Degradation Test of Polymeric Films by
Rat Cecal Contents
After showing that a chemically reduced polymer
is more soluble then its parent unreduced poly-
mer, we turned to test the combination of
reducibility and solubility. Polymers EL-7-2 and
EL-7-3 which showed the most promising char-
acteristics were examined. In this experiment, the
reduction and consequent dissolving of the poly-
meric films was tested together. Polymeric films of
EL-7-2 and EL-7-3 were cast on frosted glass
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