Synthesis and in vitro/in vivo evaluation of 5-aminosalicyl-glycine as a colon-specific prodrug of 5-aminosalicylic acid

Article abstract of DOI:10.1002/(SICI)1520-6017(200005)89:5<594::AID-JPS5>3.0.CO;2-8

A simple synthetic route for the preparation of amino acid conjugate of 5-aminosalicylic acid (5-ASA) was exploited and prepared 5-aminosalicyl-glycine (5-ASA-Gly) in good yield. In vitro and in vivo properties of 5-ASA-Gly as a colon-specific prodrug of 5-ASA were investigated using rats as the test animal. Incubation of 5-ASA-Gly with cecal or colonic contents at 37°C released 5-ASA in 65 or 27% of the dose in 8 h, respectively. No 5-ASA was detected from the incubation of 5-ASA-Gly with the homogenates of stomach or small intestine. Plasma concentration of 5-ASA-Gly decreased rapidly after intravenous administration of 5-ASA-Gly, and no 5-ASA was detected in the blood, which indicated 5-ASA-Gly was not degraded in the plasma. After oral administration of 5-ASA-Gly, about 50% of the administered dose was recovered as 5-ASA and N-acetyl-ASA and 3% as 5-ASA-Gly from feces and 14% as 5-ASA-Gly and 28% as 5-ASA and N-acetyl-ASA from urine in 24 h. These results suggested that a large fraction of 5-ASA-Gly was delivered to the large intestine and activated to liberate 5-ASA. For comparison, total recovery of 5-ASA and N-acetyl-5-ASA from feces after oral administration of 5-ASA-Gly was greater than that from sulfasalazine, which is one of the most commonly prescribed prodrugs of 5-ASA. (C) 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Full text of DOI:10.1002/(SICI)1520-6017(200005)89:5<594::AID-JPS5>3.0.CO;2-8

College of Pharmacy, Pusan National University, Pusan 609-735, Korea
Received 25 August 1999; revised 10 December 1999; accepted 23 December 1999
A simple synthetic route for the preparation of amino acid conjugate of
5-aminosalicylic acid (5-ASA) was exploited and prepared 5-aminosalicyl-glycine (5-
ASA-Gly) in good yield. In vitro and in vivo properties of 5-ASA-Gly as a colon-specific
prodrug of 5-ASA were investigated using rats as the test animal. Incubation of 5-ASA-
Gly with cecal or colonic contents at 37°C released 5-ASA in 65 or 27% of the dose in 8
h, respectively. No 5-ASA was detected from the incubation of 5-ASA-Gly with the
homogenates of stomach or small intestine. Plasma concentration of 5-ASA-Gly de-
creased rapidly after intravenous administration of 5-ASA-Gly, and no 5-ASA was
detected in the blood, which indicated 5-ASA-Gly was not degraded in the plasma. After
oral administration of 5-ASA-Gly, about 50% of the administered dose was recovered as
5-ASA and N-acetyl-ASA and 3% as 5-ASA-Gly from feces and 14% as 5-ASA-Gly and
28% as 5-ASA and N-acetyl-ASA from urine in 24 h. These results suggested that a
large fraction of 5-ASA-Gly was delivered to the large intestine and activated to liberate
5-ASA. For comparison, total recovery of 5-ASA and N-acetyl-5-ASA from feces after
oral administration of 5-ASA-Gly was greater than that from sulfasalazine, which is
one of the most commonly prescribed prodrugs of 5-ASA. © 2000 Wiley-Liss, Inc. and the
American Pharmaceutical Association J Pharm Sci 89:594–602, 2000
Keywords: colon-specific prodrug of 5-aminosalicylic acid; 5-aminosalicylic acid; co-
lon-specific delivery
ential site of absorption considering the low level
of endogenous enzymes and the long transit
time.1–6 Delayed absorption by colonic targeting
may also be useful for the treatment of diseases
susceptible to diurnal rhythm such as asthma,
gastric ulcer, or arthritis, which may have peak
symptoms at bedtime.
Inflammatory bowel diseases (IBD) such as
Crohn’s disease and ulcerative colitis have not
been rare among Europeans and Americans. The
exact cause of the IBD is not clearly understood
yet, and corticosteroids and salicylates are fre-
quently used therapeutic agents to relieve symp-
toms of such ailments.7 5-ASA is an active ingre-
dient of agents used for the long-term mainte-
nance therapy to prevent relapses of Crohn’s
Delivery of orally administered drugs specifically
to the colon is highly desirable for the efficient
treatment of diseases developed locally at the co-
lon such as ulcerative colitis, Crohn’s disease,
constipation, or colorectal cancer. Being delivered
specifically to the site of action with limited sys-
temic absorption, only a small dose is needed,
which subsequently results in reduced side ef-
fects. For orally administered therapeutic pep-
tides and proteins, the colon could be the prefer-
Correspondence to: Young Mi Kim. (E-mail: ymikim@
hyowon.pusan.ac.kr)
Journal of Pharmaceutical Sciences, Vol. 89, 594–602 (2000)
© 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association
594
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5-ASA-GLY AS COLON-SPECIFIC PRODRUG OF 5-ASA
595
disease and ulcerative colitis. Unless it is formu-
lated as sustained-release preparations or pro-
drugs for colon-specific delivery, 5-ASA is not ad-
equate to be used as such because it is absorbed
rapidly and extensively through the upper intes-
tine before it reaches to the colon.8 In addition,
systemically absorbed 5-ASA is reported to induce
nephrotic syndrome.9
tact form. Once delivered to the colon, it might
degrade by the microbial enzyme to release 5-ASA
and the amino acid. The released 5-ASA in the
large intestine should be available for the local
action.
In this study, 5-ASA-Gly was designed as a co-
lon-specific prodrug of 5-ASA. A simple synthetic
route for the preparation of amino acid conjugate
of 5-ASA in good yield is introduced. In vitro and
in vivo properties of 5-ASA-Gly as a colon-specific
prodrug of 5-ASA were evaluated.
Prodrugs aiming at the delivery of 5-ASA to the
colon have been introduced that use polymers10–12
or highly hydrophilic small molecules13–16 as
carriers. Coupling of 5-ASA to the carrier mol-
ecule achieved most commonly by means of azo
bond as exemplified by sulfasalazine (5-[[4-(2-
pyridylsulfamoyl)phenyl]azo]salicylic acid) or ol-
salazine (disodium 3,3Ј-azobis(6-hydroxybenzo-
ate)) is based on the fact that azo bonds are
readily dissociated by reduction in the large in-
testine. Glycosides14 or amide bonds16 are also
applied for attaching 5-ASA to the carrier mol-
ecule because these bonds are also dissociated in
the large intestine mostly by the microbial action.
To be colon-specific, a prodrug of 5-ASA should
be chemically and biochemically stable and non-
absorbable in the upper intestine so that it could
be delivered to the colon in intact form. And the
linkage between the drug and the promoiety
should be dissociated to liberate the active drug in
the colon.
N-acyl amide bond derived from an aromatic
carboxylic acid and an amino acid is reported to
be chemically and biochemically stable in the up-
per intestine and dissociated in the large intes-
tine by the microbial action.17 It is also reported
that p-aminohippuric acid dissociated into p-
aminobenzoic acid and glycine when it was incu-
bated with rat cecal contents.18 Glycine or taurine
conjugates of bile acids are known to undergo en-
terohepatic circulation,19 which implies that the
amide bond between the carboxyl group of a bile
acid and the amino group of glycine or taurine is
microbially degraded in the intestine.
5-Nitrosalicylic acid (5-NSA), glycine methyl es-
ter hydrochloride, N,NЈ-dicyclohexylcarbodiimide
(DCC), and 10% Pd/C were purchased from Sigma
Chemical Co. (St. Louis, MO) and used as re-
ceived. Solvents for NMR and HPLC were ob-
tained from Merck Inc. (Damstadt, Germany). All
other chemicals were reagent grade, commer-
cially available products. IR spectra were re-
corded on a Bomem MB 100 FT-IR spectropho-
tometer (Bomem).1 H-NMR spectra were taken on
a Brucker AC-200 spectrometer (Brucker), and
the chemical shifts are in ppm down field from
tetramethylsilane. Melting points were taken on
a Mel Tem II (Laboratory Devices, Holliston, MA,
USA) and were uncorrected. A Parr 4562 pressure
reactor (Parr Instrument Company, Moline, IL,
USA) was used for catalytic hydrogenation. An
Eyela Mazela-Z tissue homogenizer (Tokyo Ri-
kakikai Co., LTD., Tokyo, Japan) was used for
homogenization of the gastrointestinal tracts of
rats. A Hanil Supra K-22 centrifuge (Hanil In-
strument, Seoul, Korea) was used for centrifuga-
tion. TLCs were performed on Merck Kieselgel 60
F254, and RP-8 F254s (Merck, Damstadt, Ger-
many). Open-column chromatography and low-
pressure chromatography were performed on
Merck silica gel (70 230 mesh and 230 400
mesh), and Merck Lichroprep RP-8 size B (230
400 mesh) column, respectively. The HPLC sys-
tem consisted of Model 305, 306 pumps, a 117
variable UV detector, a Model 234 autoinjector, a
Model 805 manometric module, and a Model 811C
dynamic mixer from Gilson (Middleton, WI,
USA).
If an amino acid conjugate of 5-ASA is synthe-
sized, the mechanism of absorption might be
changed in comparison with 5-ASA and the ab-
sorption by paracellular or carrier-mediated
mechanisms might be restricted. If the compound
is hydrophilic, transcellular absorption by way of
lipid membrane permeation might be limited in
the upper intestine. Because the amide bond of
N-aromatic acyl-amino acid conjugate is reported
to be stable in the upper intestine,17 a large frac-
tion of orally administered N-5-aminosalicyl-
amino acid might be delivered to the colon in in-
A male Sprague-Dawley rat was anesthetized by
diethyl ether, and a midline incision was made
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596
JUNG, LEE, AND KIM
and various segments of the gastrointestinal tract
were obtained. The tissue and contents of the
stomach and small intestine and the contents of
the cecum or colon were homogenized and diluted
with isotonic phosphate buffer (pH 6.8) to the con-
centration of 10 w/v%. To each 100 ␮L portion of
the homogenate, 10, 50, 100, or 200 ␮L portion of
the solution of 5-ASA, 5-ASA-Gly, or N-acetyl-5-
ASA (50␮g equivalent of 5-ASA/mL) in isotonic
phosphate buffer (pH 6.8) and an appropriate
amount of methanol were added to make the final
volume of 1 mL. The standard solutions in con-
centration of 0.5, 2.5, 5.0, or 10.0␮g 5-ASA/mL (or
equivalent) were obtained by the preceding pro-
cesses. A calibration curve for 5-ASA, 5-ASA-Gly,
or N-acetyl-5-ASA was constructed from the con-
centration of the standard solution versus the
peak area on HPLC, which was determined by the
following procedure. Standard or blank solution
(1 mL) was mixed on a vortex mixer for 2 min,
centrifuged at 10,000 × g for 5 min, and filtered
through a membrane filter (0.45 ␮m). The filtrate
(20 ␮L) was injected on a Synchropac ODS col-
umn (250 × 4.6 mm, 5 ␮m) (Gilson) and eluted
with the mobile phase at a flow rate of 1.5 mL/min
and at a pressure of about 2000 psi. The mobile
phase consisted of 10% methanol in phosphate
buffer (pH 6.0) containing 0.5 mM tetrabutylam-
monium chloride, which was filtered through 0.45
␮m membrane filter before use. The eluate was
monitored by measuring the absorption at 254 nm
at sensitivity of AUFS 0.01. The Gilson 712 soft-
ware was used for the data analysis. The reten-
tion time of 5-ASA, N-acetyl-5-ASA, or 5-ASA-Gly
was 280 s, 820 s, or 370 s, respectively. Concen-
tration of 5-ASA, 5-ASA-Gly, or N-acetyl-5-ASA
in the sample was determined from the calibra-
tion curve.
under reduced pressure. The residue was loaded
on a silica gel open column and eluted with
CHCl3/MeOH (100/1.5), from which 5-nitrosali-
cyl-glycine methyl ester was obtained (4.4 g, 65%
yield). mp: 140
142°C; IR (nujol) ␯max(CסO):
1
1,635 cm−1, 1,747 cm−1, H NMR (CDCl3): 3.8 (s,
3H, COOCH3), 4.9 (d, 2H, NHCH2CO), 7.0 8.8
(m, 3H, ArH).
5-Nitrosalicyl-glycine methyl ester (1 g, 3.95
mmol) and 200 mg of 10% Pd/C in methanol (20
mL) was placed in a Parr pressure reactor and
hydrogenated at 50 psi for 1 h. After filtering and
removal of methanol, 1N NaOH (30 mL) was
added and reacted for 5 h under nitrogen. On ad-
justment of pH to 3 4, 5-ASA-Gly (0.69 g, 83%
yield) was obtained as white precipitates. mp:
297°C (decomp); IR (nujol) ␯max(CסO): 1,620
cm−1, 1,648 cm−1; 1H NMR (DMSO-d6): 3.9 (d, 2H,
NHCH2CO), 6.6 7.0 (m, 3H, ArH).
The apparent partition coefficients were deter-
mined as follows. To a solution (10 mL) of 5-ASA
(100 (g/mL) or 5-ASA-Gly (100 ␮g equivalent of
5-ASA/mL) in pH 6.8 isotonic phosphate buffer,
10 mL of chloroform (or n-octanol) saturated with
pH 6.8 isotonic phosphate buffer was added. The
mixture was shaken for 10 h and left for 4 h at
37°C. Concentration of 5-ASA or 5-ASA-Gly in the
aqueous phase was analyzed by HPLC. The ap-
parent partition coefficients were calculated by
using the equation (Co − Cw)/Cw, where Co and Cw
represent the initial and equilibrium concentra-
tion of the drug in aqueous phase, respectively.
The pH stability was determined by preparing
the solution of 5-ASA-Gly (140 (g/mL) in pH 1.2
hydrochloric acid buffer or pH 6.8 phosphate
buffer and incubating the solution at 37°C for 6 h.
At a predetermined time interval, a 20 ␮L portion
of the solution was removed and analyzed the con-
centration of 5-ASA by HPLC.
To the solution of 5-nitrosalicylic acid (5 g, 27.3
mmol) in 170 mL of anhydrous ethyl acetate, DCC
(6.2 g, 30.0 mmol) was added in portions with stir-
ring at 0°C for 1 h. To the reaction mixture, gly-
cine methyl ester (2.4 g, 27.3 mmol) was added
and stirred mechanically for 3 h at 0°C and 72 h
at room temperature. After filtration, the filtrate
was evaporated under reduced pressure to re-
move the solvent. The oily residue thus obtained
was extracted with a saturated solution of
NaHCO3. The combined extract was acidified
with 3N HCl, extracted with ethyl acetate, dried
with anhydrous Na2SO4, and the solvent removed
A male Sprague-Dawley rat was anesthetized by
diethyl ether and a midline incision was made.
Sections of stomach and small intestine were col-
lected separately, homogenized, and the homog-
enate was diluted to half concentration with iso-
tonic acetate buffer (pH 4.5) for stomach and with
isotonic phosphate buffer (pH 6.8) for small intes-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000

5-ASA-GLY AS COLON-SPECIFIC PRODRUG OF 5-ASA
597
tine. We adopted the pH values for stomach and
small intestine as 4.5 and 6.8, respectively, which
were the reported pH values normally found in
the rat gastrointestinal tract.20–21 To a 0.2-g por-
tion of each homogenate, 0.8 mL of 5-ASA-Gly
solution in pH 6.8 isotonic phosphate buffer (140
␮g equivalent of 5-ASA) was added and the mix-
ture was incubated for 6 h at 37°C. At appropriate
time intervals, the sample was centrifuged at
5,000 rpm for 3 min. To a 0.1-mL portion of the
supernatant, 0.9 mL of methanol was added, vor-
texed for 2 min, and centrifuged for 5 min at
10,000 × g. The concentration of 5-ASA or N-ace-
tyl-5-ASA in a 20 ␮L portion of the supernatant
was determined by HPLC as described previ-
ously.
portion of the supernatant was analyzed by
HPLC as described previously.
Male Sprague-Dawley rats had been starved for
24 h except for water. They were anesthetized
with diethyl ether and cannulated with polyeth-
ylene tubing SP 45 (0.96 mm o.d.; Natsum, Japan)
through the femoral artery. 5-ASA or 5-ASA-Gly
(50 mg equivalent of 5-ASA/kg), which was dis-
solved in 1.0 mL of physiologic saline solution by
adding minimum amount of 1N-NaOH, was ad-
ministered by an intragastric probe. The same
protocol was carried out for the sample collection
and analysis of 5-ASA-Gly, 5-ASA, and N-acetyl-
5-ASA in the blood as described previously.
The cecal and colonic segments of the intestine
were cut open and their contents were collected
separately in a glove box, which was previously
displaced by nitrogen. To a 0.1-g portion of the gut
contents, 0.9 mL of 5-ASA-Gly solution in pH 6.8
isotonic phosphate buffer (140 ␮g equivalent of
5-ASA) was added and the mixture was incubated
at 37°C. At appropriate time intervals, the
sample was taken out and centrifuged at 5,000
rpm for 3 min. To a 0.1-mL portion of the super-
natant, 0.9 mL of methanol was added, vortexed
for 2 min, and centrifuged for 5 min at 10,000 × g.
The concentration of 5-ASA or N-acetyl-5-ASA in
a 20 ␮L portion of the supernatant was deter-
mined by HPLC as described previously.
Male Sprague-Dawley rats were placed in a meta-
bolic cage and starved for 24 h before use for the
experiments but had free access to water. 5-ASA,
5-ASA-Gly, or sulfasalazine (50-mg equivalent of
5-ASA/kg) was orally administered as described
previously. The fecal and urinary samples were
collected separately at 2-h intervals and stored
immediately in a freezer before being analyzed by
the following procedure. The fecal or urinary
sample was diluted with isotonic phosphate
buffer solution (pH 6.8) to 100-fold or 10-fold, re-
spectively. The sample was vortexed and centri-
fuged at 5,000 rpm for 3 min. To a 0.1-mL portion
of the supernatant, 0.9 mL of methanol was
added, vortexed for 2 min, and centrifuged for 5
min at 10,000 × g. The concentration of 5-ASA,
N-acetyl-5-ASA, or 5-ASA-Gly in a 20 ␮L portion
of the supernatant was determined by HPLC as
described previously.
Male Sprague-Dawley rats weighting 200 250 g
were anesthetized with diethyl ether and cannu-
lated with polyethylene tubing SP 45 (0.96 mm
o.d.; Natsum, Japan) through the femoral artery,
and administered 10 mg equivalent of 5-ASA/kg
dissolved in 4.0 mL of physiologic saline solution
through the femoral vein. Blood samples were col-
lected from the femoral artery with a heparinized
syringe at appropriate time intervals and centri-
fuged at 5,000 rpm for 3 min. To the 0.1-mL por-
tion of the separated plasma, 0.9 mL of methanol
was added. It was vortexed for 2 min and centri-
fuged at 10,000 × g for 5 min. The concentration of
5-ASA-Gly, 5-ASA, or N-acetyl-5-ASA in a 20-␮L
Preparation of 5-ASA-Gly was achieved as shown
in Scheme 1: (i) 5-NSA was reacted with glycine
methyl ester in the presence of DCC to produce
5-NSA-Gly methyl ester. (ii) Reduction of nitro
group with 10% Pd/C gave 5-aminosalicyl-glycine
methyl ester (iii), and hydrolysis of the ester af-
forded 5-ASA-Gly (iv). The overall yield was high,
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 5, MAY 2000

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JUNG, LEE, AND KIM
Scheme 1. Synthesis of 5-aminosalicyl-glycine from
5-nitrosalicylic acid.
and the reaction processes were relatively simple
compared with the method using 5-ASA as a
starting material.15
Figure 1. Release profiles of 5-ASA during incuba-
tion of 5-ASA-Gly with homogenates of various seg-
ments of the gastrointestinal tract of rats at 37°C.
5-ASA-Gly (equiv. to 140␮g of 5-ASA) in 1.0 mL of ten-
fold dilution of (᭹) cecal contents, (᭺) colonic contents,
(᭿) stomach and small intestinal tissue/contents in iso-
tonic phosphate buffer (pH 6.8). Data are mean SE
(n ס 5).
Apparent partition coefficient of 5-ASA, which
was determined at 37°C using chloroform/ phos-
phate buffer (pH 6.8) or n-octanol/phosphate
buffer (pH 6.8), was 0.03 or 0.04, respectively, and
that of 5-ASA-Gly was 0.18 or 0.24, respectively.
Because 5-ASA possesses a low partition coeffi-
cient, it is absorbed in the upper intestine by way
of paracellular pathways or carrier-mediated
transport rather than transcellular passive diffu-
sion.25 In the case of 5-ASA-Gly, absorption by
transcellular passive diffusion might be limited
because of the low partition coefficient, which
may result in limited overall absorption in the
upper intestine, unless it is absorbed by the same
mechanism as 5-ASA.
Chemical stability of 5-ASA-Gly was deter-
mined at 37°C in pH 1.2 hydrochloric acid buffer
or pH 6.8 phosphate buffer solution. No 5-ASA
was detected during the 6 h of the incubation pe-
riod, which indicated that 5-ASA-Gly might be
chemically stable during the transit through the
gastrointestinal tract.
uted in the mucosal membranes of the small in-
testine, in vitro experiments to determine the re-
lease of 5-ASA after incubation of 5-ASA-Gly with
the homogenate of stomach or small intestine (tis-
sue and contents) of rats were carried out at 37°C.
Incubation of 5-ASA-Gly with the cecal and co-
lonic contents of rats was also carried out at 37°C,
and the results are shown in Figure 1. On incu-
bation of 5-ASA-Gly with the homogenate of
stomach or small intestine at 37°C, 5-ASA was
not detected, which indicated that the prodrug
might be stable against peptidases in the upper
intestine. When 5-ASA-Gly was incubated with
the cecal contents, about 40% of the dose was re-
leased as 5-ASA in 2 h and about 65% in 8 h. With
the colonic contents, the fraction of the dose re-
leased as 5-ASA was only 27% in 8 h, which indi-
cated that prodrug activation took place most
readily in the rat cecum, where the bacterial
counts are high as in the human colon. N-acetyl-
5-ASA, a major metabolite of 5-ASA, was not de-
tected, which confirms the early findings that
colonocytic acetylation is responsible for the
acetylation of the aromatic amino group rather
than bacterial system in the intestine.8,17
To investigate the hydrolysis of 5-ASA-Gly by the
peptidases, which are known to be highly distrib-
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5-ASA-GLY AS COLON-SPECIFIC PRODRUG OF 5-ASA
599
Figure 2. Plasma concentration profiles of 5-ASA-
Gly, 5-ASA, and N-acetyl-5-ASA after intravenous ad-
ministration of 5-ASA-Gly (10 mg eqv. of 5-ASA/kg)
through the femoral vein of rats. Blood was collected at
predetermined interval from a cannulated femoral ar-
tery. Data are mean SE (n ס 5).
Figure 3. Plasma concentration profile of 5-ASA and
N-acetyl-5-ASA after oral administration of 5-ASA (50
mg/kg) in rats. Blood was collected at predetermined
interval from a cannulated femoral artery. Data are
mean SE (n ס 5).
level of 5-ASA-Gly in the plasma was extremely
low throughout the whole experimental period (24
h), which might indicate the limited oral absorp-
tion and the facile elimination of 5-ASA-Gly from
the plasma as illustrated in Figure 2. The plasma
concentration profile of 5-ASA or N-Ac-5-ASA
showed tmax around 2 h, which seemed shorter
than usually expected. It is reported that the
transit time through the upper intestine of the rat
is about the same or slightly shorter than that of
the man.23 The transit time of a tablet (7 mm in
diameter) to the large intestine taken with a
regular meal is reported to be 1.5 h in man.24 It
might be shortened if the drug were administered
as a solution form in fasting state as we did in this
experiment. In this experimental condition, the
transit time through the upper intestine might
have been shortened. This might explain the
short tmax value for 5-ASA as shown in Figure 3.
Shortened transit time through the upper intes-
tine and the facile hydrolysis in the cecum might
be responsible for the early detection of 5-ASA
and N-acetyl-5-ASA in the plasma or the short
tmax values observed after oral administration of
5-ASA-Gly. The levels of 5-ASA and N-acetyl-5-
ASA in the plasma were very low for the whole
experimental period (24 h), which differs greatly
from the case in which the equivalent amount of
After intravenous administration of 5-ASA-Gly,
plasma concentration of 5-ASA-Gly, 5-ASA, or
N-acetyl-5-ASA was determined by HPLC and
the results are shown in Figure 2. Plasma concen-
tration of 5-ASA-Gly decreased rapidly and
5-ASA or N-acetyl-5-ASA was not detected, indi-
cating that 5-ASA-Gly was not degraded in the
blood and eliminated rapidly.
5-ASA or 5-ASA-Gly was administered orally (50
mg equivalent of 5-ASA/kg) and plasma concen-
tration profiles of 5-ASA and N-acetyl-5-ASA
were determined and compared. Absorption of
5-ASA took place rapidly (tmax Յ 0.5 h), showing
the tmax is shorter than the reported values of 1
h.22 Plasma concentration of N-acetyl-5-ASA was
higher than that of 5-ASA and elimination was
nearly complete in 7 h (Fig. 3).
After oral administration of 5-ASA-Gly,
plasma concentration of 5-ASA-Gly, 5-ASA, or
N-acetyl-5-ASA was determined (Fig. 4). The
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JUNG, LEE, AND KIM
Figure 5. Percent recovery of 5-ASA and N-acetyl-5-
ASA in feces and urine after oral administration of (A)
5-ASA, (B) 5-ASA-Gly, and (C) sulfasalazine (50 mg
eqv. of 5-ASA/kg) in rats. Data are mean SE (n ס 5).
tively. From urine, recovery (% of the dose) of
5-ASA for 5-ASA-Gly, 5-ASA, or sulfasalazine
was 6.5, 16.0, or 7.0, respectively, and that of
N-acetyl-5-ASA for 5-ASA-Gly, 5-ASA, or sul-
fasalazine was 22.0, 64.0, or 23.0, respectively.
Recovery (% of the dose) of 5-ASA-Gly from feces
or urine was 3.5 or 14.0 (not shown in Fig. 5),
respectively.
Figure 4. Plasma concentration profiles of 5-ASA-
Gly, 5-ASA, and N-acetyl-5-ASA after oral administra-
tion of 5-ASA-Gly (50 mg eqv. of 5-ASA/kg) in rats.
Blood was collected at predetermined interval from a
cannulated femoral artery. Data are mean
SE
(n ס 5).
Recently, Zhou et al.25 reported that a fraction
of 5-ASA absorbed in the intestinal cell is metabo-
lized to N-acetyl-5-ASA, which is secreted into the
intestinal lumen predominantly. The fraction of
5-ASA absorbed transcellularly and not metabo-
lized or transported paracellularly enters the gen-
eral circulation where it is metabolized in the
liver or kidney and finally excreted through urine
as an intact drug and metabolite. They reasoned
that metabolite levels in the urine represent sys-
temic or hepatic metabolism, and luminal and fe-
cal metabolite levels represent a measure of in-
testinal metabolism. Therefore, the fraction of
N-acetyl-5-ASA in the lumen or feces might be
related to therapeutic effect, whereas that in the
urine might be relate to side effects.
free 5-ASA was administered orally (Fig. 3). Be-
cause 5-ASA-Gly was not degraded to release
5-ASA in the plasma (Fig. 2), or it did not released
5-ASA on incubation with the upper intestinal
constituents (Fig. 1), 5-ASA or N-acetyl-5-ASA
appearing in the plasma might originate from the
one absorbed in the large intestine. Activated by
the microbial enzyme in the large intestine,
5-ASA-Gly releases 5-ASA at a controlled rate,
and a portion of the released 5-ASA might be ab-
sorbed in the large intestine and appears in the
plasma for an extended period of time.
In summary, only about 7% of the dose was
recovered as N-acetyl-5-ASA from the feces and
about 80% of the dose was recovered as 5-ASA
and N-acetyl-5-ASA from the urine in 24 h with
free 5-ASA. With 5-ASA-Gly, about 21% of the
dose was recovered as N-acetyl-5-ASA from the
feces and about 28% of the dose was recovered as
5-ASA and N-acetyl-5-ASA from the urine in 24 h.
With sulfasalazine, 13% of the dose was recovered
as N-acetyl-5-ASA from the feces and 30% of the
dose was recovered as 5-ASA and N-acetyl-5-ASA
from the urine in 24 h.
Concentrations of 5-ASA and N-acetyl-5-ASA in
the feces and urine 24 h after oral administration
of 5-ASA-Gly, 5-ASA, or sulfasalazine were deter-
mined and the results are shown in Figure 5. Re-
covery (% of the dose) of 5-ASA from the feces for
5-ASA-Gly, 5-ASA, or sulfasalazine was 31.0, 0.3,
or 24.0, respectively. Recovery (% of the dose) of
N-acetyl-5-ASA from feces for 5-ASA-Gly, 5-ASA,
or sulfasalazine was 20.8, 7.0, or 13.3, respec-
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5-ASA-GLY AS COLON-SPECIFIC PRODRUG OF 5-ASA
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