Synthesis and evaluation of sulfate conjugated metronidazole as a colon-specific prodrug of metronidazole

Article abstract of DOI:10.3109/1061186X.2011.639024

For an effort to improve therapeutic property of metronidazole (MTZ) which is a drug of choice for protozoal infections such as luminal amoebiasis, sulfate conjugated metronidazole (MTZS) was prepared and evaluated as a colon-specific prodrug of MTZ. The apparent partition coefficient of MTZ was greatly reduced by the sulfate conjugation. While (bio)chemically stable in the contents of the upper intestine, MTZS was rapidly cleaved to liberate MTZ on incubation with the cecal contents of rats. MTZ liberated from MTZS metabolized quickly at least partly by a microbial nitroreductase, suggesting the relevance of the metabolism to bioactivation of MTZ for antimicrobial action. Consistent with the hypothesis, MTZS elicited antibacterial activity in the cecal contents, which was as potent as free MTZ. The systemic absorption of MTZS was very low after oral administration of MTZS. In parallel with this, whereas MTZ disappeared mostly during the transit of the proximal small intestine, a substantial amount of MTZS remained in the small intestine, moving down to the large intestine where it metabolized rapidly. In addition to the efficient colonic delivery of MTZS, MTZS markedly reduced the systemic absorption of MTZ. Taken together, MTZS may be a potential colon-specific prodrug of MTZ which possesses improved therapeutic and toxicological properties.

Full text of DOI:10.3109/1061186X.2011.639024

Journal of Drug Targeting, 2012; 20(3): 255–263
© 2012 Informa UK, Ltd.
ISSN 1061-186X print/ISSN 1029-2330 online
DOI: 10.3109/1061186X.2011.639024
RESEARCH ARTICLE
Synthesis and evaluation of sulfate conjugated metronidazole
as a colon-specific prodrug of metronidazole
Hyunjeong Kim1, Yonghyun Lee1, Hansun Yoo1, Jihye Kim1, Hyesik Kong2, Jeong-Hyun Yoon1,
Yunjin Jung1, and Young Mi Kim1
1Laboratory of Biomedicinal/Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan, Korea and
2Urologic Oncology Branch, National Cancer Institute, Bethesda, MD, USA
Abstract
For an effort to improve therapeutic property of metronidazole (MTZ) which is a drug of choice for protozoal infections
such as luminal amoebiasis, sulfate conjugated metronidazole (MTZS) was prepared and evaluated as a colon-specific
prodrug of MTZ. The apparent partition coefficient of MTZ was greatly reduced by the sulfate conjugation. While (bio)
chemically stable in the contents of the upper intestine, MTZS was rapidly cleaved to liberate MTZ on incubation with
the cecal contents of rats. MTZ liberated from MTZS metabolized quickly at least partly by a microbial nitroreductase,
suggesting the relevance of the metabolism to bioactivation of MTZ for antimicrobial action. Consistent with the
hypothesis, MTZS elicited antibacterial activity in the cecal contents, which was as potent as free MTZ. The systemic
absorption of MTZS was very low after oral administration of MTZS. In parallel with this, whereas MTZ disappeared
mostly during the transit of the proximal small intestine, a substantial amount of MTZS remained in the small intestine,
moving down to the large intestine where it metabolized rapidly. In addition to the efficient colonic delivery of MTZS,
MTZS markedly reduced the systemic absorption of MTZ. Taken together, MTZS may be a potential colon-specific
prodrug of MTZ which possesses improved therapeutic and toxicological properties.
Keywords: Controlled drug delivery, drug delivery, drug targeting, oral drug delivery
Introduction
the treatment of inflammatory bowel disease (Egan and
Intestinal amebiasis, an infectious disease caused by
Entamoeba histolytica, a single-celled protozoan para-
site, is one of major public health problems in developing
countries. e trophozoite form of E. histolytica invades
the colonic epithelium and causes consequent amebic
colitis or amebic abscesses in the brain, which are dread-
ful complications of E. histolytica infection(Stanley,
2003). World Health Organization (WHO), in its recent
estimates, has placed the death toll from amebiasis at
40,000–100,000 lives annually (Petri, 2003).
Metronidazole (MTZ), an antibiotic of which chemi-
cal structure is classified as nitro 5-imidazoles, is a
drug of choice for the treatment of intestinal amebiasis
and other anaerobic protozoal and bacterial infections
(Freeman et al., 1997). In addition, MTZ is being used for
Sandborn, 1998). Pharmacologically, MTZ enters micro-
bial cells and goes through the reduction of the nitro
group of it by low redox potential electron transport pro-
teins. A product generated by this process forms a cova-
lent adduct with guanine or cytosine in the DNA strand
and/or functionally essential proteins in the microbes,
resulting in antimicrobial effects (Ludlum et al., 1988;
Leitsch et al., 2007).
e pharmacokinetic profile of MTZ indicates that
greater than 90% of the dose is rapidly absorbed after
oral administration with conventional tablet formula-
tion (Lau et al., 1992; Lamp et al., 1999). As a result, the
amount of MTZ that reach the large intestine where
trophozoites reside is minimal. Although small amount
of MTZ reached the target site can still promote the
Address for Correspondence: Professor Young Mi Kim, College of Pharmacy, Pusan National University, Busan, Korea 609-735.
Tel: 051-510-2807. Fax: 051-513-6754. E-mail: ymikim@pusan.ac.kr Dr. Yunjin Jung, College of Pharmacy, Pusan National University, Busan,
Korea 609-735. Tel: 051-510-2527. Fax: 051-513-6754. E-mail: jungy@pusan.ac.kr
(Received 16 June 2011; accepted 23 October 2011)
255

256 H. Kim et al.
elimination of amebiasis, it is not without unwanted
systemic adverse effects such as nausea, vomiting, head-
ache, glossitis, and in few cases convulsions (Kapoor
et al., 1999). For these reasons, it is an attractable
therapeutic strategy to deliver MTZ specifically to the
infected target site, maximizing therapeutic efficacy
and minimizing adverse effects attributed to systemi-
cally absorbed drug. erefore, researches on colonic
delivery of MTZ with pharmaceutical and prodrug
approaches have been widely carried out (Krishnaiah
et al., 2001; Krishnaiah et al., 2002; Mundargi et al.,
2007; Kim et al., 2009).
In designing colon-specific prodrug, the drug mole-
cule is coupled with a promoiety such as non-absorbable
polymers or hydrophilic small molecules by a linkage
which is stable in the upper intestine and labile in the
colon. As a result, absorption of drug-promoiety com-
pound in the upper intestine is very limited and a sub-
stantial amount of it is delivered to the colon, where an
active drug is released by enzymes produced by intesti-
nal microorganisms (Sinha and Kumria, 2001; Jung and
Kim, 2010).
Van Eldere et al. reported on the isolation and iden-
tification of intestinal steroid-desulfating bacteria from
rats and humans (Van Eldere et al., 1988). Huijghebaert
et al. reported that microbial estrone sulfatase activity
in the intestinal contents of rats was high in the cecum,
but very low in the small intestine (Huijghebaert et al.,
1984). ese reports suggested that sulfatase in the gas-
trointestinal tract is of microbial origin and its activity
is high in the cecum, but very low in the small intestine.
erefore, it is very likely that sulfate moiety satisfies
the aforementioned requirement for a colon-specific
promoiety. In fact, we previously reported that sulfated
steroids, prednisolone and dexamethasone 21-sulfate
sodium, act as a colon-specific prodrug of the steroids in
vivo and in vitro (Jung et al., 2003; Kim et al., 2006). In this
study, sulfate moiety was introduced as a colon-specific
promoiety for colonic delivery of MTZ, which should pro-
vide another chemical option for colonic delivery of the
anti-amebic agent using a prodrug approach. Recently, a
colon-specific prodrug of MTZ has been reported, where
N-nicotinoyl-(2-acetoxy)-D,L-glycine is adopted as a
colon-specific promoiety (Kim et al., 2009). Sulfate con-
jugated MTZ (MTZS) was prepared in high yield, which
is an advantage for industrialization. e antimicrobial
activity in the cecal contents and biodisposition of MTZS
were investigated to evaluate the conjugate as a colon-
specific prodrug of MTZ with improved therapeutic and
toxicological properties.
other chemicals were reagent grade, commercially avail-
able products. IR spectra were recorded on a Bomem
MB 100 FT-IR spectrophotometer (Bomem, Ontario,
1
Canada). H-NMR spectra were taken on a Varian AS
500 spectrometer (Santa Clara, CA) and the chemical
shifts were in ppm downfield from tetramethylsilane.
Elemental analysis was carried out by an Elemental
Analyzer System (Profile HV-3, Manchester, UK). A
Polytron PT 3100 homogenizer (Kinematica, Bohemia,
NY) was used for homogenization of the tissue of the
gastrointestinal tracts of rats. A Taitec microincubator
M-36 (Tokyo, Japan) was used for incubation. e ani-
mal protocol used in this study has been reviewed and
approved by the Pusan National University–Institutional
Animal Care and Use Committee (PNU-IACUC) on their
ethical procedures and scientific care. (Approval num-
ber: PNU-2008-0010).
Preparation of MTZS
To the solution of MTZ (1.71g, 10.0 mmol) in 64 mL of
anhydrous benzene and pyridine (1/1), STT (3.80g, 17.0
mmol) was added in portions with stirring at 56–60°C
for 20 min. e reaction mixture was evaporated under
reduced pressure to remove the solvent and obtained
metronidazole sulfate triethylammonium (MTZST) as
oily residue. After purification by silica gel open column
chromatography, MTZST was dissolved in minimum
amount of water and suspended into a solution of 10%
NaCl with mechanical stirring for 1h. e resulting pre-
cipitates, MTZS, were collected by suction filtration and
recrystallized from absolute ethanol (yield 80%). mp:
194–195 (dec). IR (nujol) cm−1: 1613, 1545, 1279, 1055,
830. 1H-NMR (D2O): 2.7 (s, 3H), 4.4 (t, 2H), 4.8 (t, 2H), 8.4
(s, 1H). EA for C6H8N3SO6Na: C, H, N, S.
HPLC analysis of drugs
e HPLC system consisted of Model 305 and 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). A symmetry
C18 column (Waters, Seoul, Korea; 4.6 × 250 mm, 5 µm)
with a guard column (Waters; 3.9 × 20 mm) was used.
e mobile phase consisted of 0.05M, pH 4.5 phosphate
buffer/acetonitrile (9/1) and was filtered through a 0.45
µm membrane filter before use. e mobile phase was
eluted at a rate of 1 mL/min. e eluate was monitored
by measuring the absorbance at 313 nm at a sensitiv-
ity of AUFS 0.01. Gilson 712 software was employed for
the data analysis. e retention time of MTZ and MTZS
was 8.7 and 6.3 min, respectively. Concentration of MTZ
and MTZS in the sample was calculated from the cali-
bration curve. e calibration curve was constructed at
the concentration range of 1–50 µg/mL. All the calibra-
tion curves were constructed by preparing the standard
samples using biological media corresponding to each
in vitro/in vivo experiment (the correlation coefficients
of the calibration curves were between 0.999–0.996). In
the case of cecal contents, the autoclaved cecal contents
Material and methods
Materials
MTZ and sulfatrioxide triethylamine complex (STT) were
purchased from Sigma Chemical Co. (St. Louis, MO) and
were used as received. Solvents for NMR and HPLC were
obtained from Merck Inc. (Darmstadt, Germany). All
Journal of Drug Targeting

A colon-specific prodrug of metronidazole 257
were used. e detection limit was about 0.5 and 0.8 µg/
mL for MTZ and MTZS, respectively, in our experimental
condition.
obtained from rats with TNBS-induced colitis or pre-
treated with 0.2 mM of dicoumarol. e whole process
was carried out in a glove box, which was previously dis-
placed by nitrogen. At an appropriate time interval, 200
µL portion of the sample was removed and centrifuged at
14,000 rpm for 3 min. To a 100 µL portion of the superna-
tant, was added 900 µL of methanol to precipitate protein
in the sample, vortexed for 2 min, and centrifuged for
5 min at 14,000 rpm. e concentration of MTZ and/or
MTZS in a 20 µL portion of the supernatant was deter-
mined by HPLC.
Apparent partition coefficient, solubility and
chemical stability
To measure solubility, MTZ or MTZS (50 mg equivalent
of MTZ) was placed in a microtube containing 1mL of pH
6.8 isotonic phosphate buffer solution. It was shaken for
24 h at 25°C. After centrifugation, a 20 µL portion of the
supernatant was analyzed by HPLC. For measurement
of apparent partition coefficients, to a 20 mL portion of
MTZ (1.0 mM) or MTZS solution (1.0 mM) in 0.1 M, pH
7.4 phosphate buffer solution saturated with 1-octanol,
was added 20 mL of 1-octanol saturated with 0.1 M, pH
7.4 phosphate buffer solution. e mixture was shaken for
24 h at 37°C. Concentration of MTZS or MTZ in the aque-
ous phase was analyzed by HPLC. e apparent partition
coefficient was calculated by using the equation (Co−Cw)
% Cw where Co and Cw represent the initial and equilib-
rium concentration, respectively, of the drug in aqueous
phase. e pH stability was determined by incubating
the solution of MTZS (1.0 mM) in pH 1.2 hydrochloric
acid buffer, pH 4.5 acetate buffer and pH 7.4 phosphate
buffer at 37°C for 24h. At a predetermined time interval, a
20 µL portion of the solution was removed and analyzed
the concentration of MTZ and MTZS by HPLC.
Antimicrobial activity of MTZS in the cecal contents
e cecal contents were diluted with Lysogeny broth (LB)
brothtoaffordthe20%(w/v)suspension. A7.5 mLportion
of MTZ or MTZS solution (equivalent to 500ppm MTZ)
in LB broth was mixed with 7.5 mL of the cecal contents
immediately followed by immersion of a dialysis tube
(Slide-A-Lyzer® MINI Dialysis Unit, Pierce, Rockford,
IL) containing E. Coli (XL1-Blue, Stratagene, Santa Clara,
CA; 200 µL, O.D.: 0.18–0.25) in each cecal suspension,
which was incubated at 37°C in a shaking incubator. All
the processes were performed in a nitrogen glove bag.
For comparison, the same experiment was done without
the cecal contents. e antimicrobial activity of MTZ and
its prodrug was determined by measurement of O.D. of
the E.Coli 8 h after incubation.
Incubation of drugs with the contents of rat
small intestine
Incubation of MTZ and MTZS with the liver
homogenates and the plasma
A male Sprague–Dawley rat (210–275 g, 7–8 weeks old)
was anesthetized by diethyl ether and a midline incision
was made. e contents of the small intestine was col-
lected and diluted to half concentration with 0.05 M, pH
6.8 isotonic phosphate buffer. A 2 mL of the contents sus-
pension was mixed with 2 mL of MTZ or MTZS solution
(200 ppm equivalent of MTZ) dissolved in pH 6.8 isotonic
phosphate buffer. e mixture was incubated at 37°C in
a shaking incubator. At an appropriate time interval, 200
µL portion of the sample was taken and centrifuged at
14,000 rpm for 3 min. To a 100 µL portion of the super-
natant, was added 900 µL of methanol to precipitate pro-
tein in the sample, vortexed for 2 min, and centrifuged
for 5 min at 14,000 rpm. e concentration of MTZ and
MTZS in a 20 µL portion of the supernatant was deter-
mined by HPLC.
To determine the metabolic stability of MTZ and MTZS
in liver, the rat liver was homogenized and diluted with
saline to 20% (w/v). A 2 mL portion of MTZ or MTZS
(200 ppm equivalent of MTZ) in pH 6.8 phosphate was
mixed with 2 mL of the 20% liver homogenate. e mix-
ture was incubated at 37°C in a shaking incubator. To
determine the metabolic stability in plasma, a 200 µL
portion of MTZ or MTZS (200ppm equivalent of MTZ) in
pH 7.4 phosphate buffer and an 800 µL portion of the rat
plasma were mixed and incubated at 37°C in a shaking
incubator. At an appropriate time interval, a 200 µL por-
tion of the liver homogenates sample was removed and
centrifuged at 14,000 rpm for 5 min. To a 100 µL portion
of the supernatant or the plasma sample, was added a 900
µL portion of methanol to precipitate protein in the sam-
ples. It was vortexed for 2 min, and centrifuged for 5 min
at 14,000 rpm. e concentration of MTZ or MTZS in a 20
µL portion of the samples was determined by HPLC.
Incubation of drugs with the cecal contents of rat
e cecal segment of the intestine was cut open and
the contents were collected. e cecal contents were
diluted with 0.05 M, pH 6.8 phosphate buffer solution
to the concentration of 20% (w/v) and filtered through
a piece of medical gauze to remove the fibrous mass. A
2 mL portion of MTZ or MTZS solution (at an appropri-
ate concentration) in 0.05 M, pH 6.8 isotonic phosphate
buffer was mixed with 2 mL of the cecal contents, which
was incubated at 37°C in a shaking incubator. In separate
experiments, MTZ was incubated with the cecal contents
Analysis of MTZ and/or MTZS in the blood and various
segments of rat gastrointestinal tract after oral
administration of drugs
Male SD rats 250–260 g were maintained with free access
to a stock diet and water. ese rats were fasted overnight
(16 h) before administration of MTZ or MTZS. Water
bottles were removed from the cages at least 30 min
before drug administration to assure that stomach would
be empty. MTZ or MTZS (4 mg equivalent of MTZ/kg) in
© 2012 Informa UK, Ltd.

258 H. Kim et al.
isotonic saline solution was orally administered to rats.
After an appropriate time interval, the animals were
sacrificed by ether anesthesia. Blood was collected by
intra-cardiac puncture through a heparinized syringe
before death. After blood collection and the contents of
various segments of gastrointestinal tract were obtained
(Kim et al., 2009). Blood samples were immediately cen-
trifuged for 5 min at 14,000 rpm and the concentration
of MTZ and/or MTZS in the plasma samples was deter-
mined by HPLC. e contents of proximal small intestine
(PSI), distal small intestine (DSI), cecum and colon were
collected separately, and the concentration of MTZ and/
or MTZS by HPLC was analyzed.
group in MTZ to afford MTZS in high yield. Structure of
MTZS was identified from IR, H-NMR spectra and ele-
1
mental analysis. Data from the elemental analysis were
in good agreement with the calculated values for C, H, N
and S (within 0.5% from the calculated values). Infrared
spectra showed two strong absorption band originating
from -S=O asymmetric and symmetric stretching of sul-
fate ester around 1279 and 1055 cm−1, respectively. e
1
most characteristic change in H-NMR spectra was the
downfield shift of the two protons on α-carbon to 0.5 ppm
by the introduction of sulfate ester group.
Apparent partition coefficient, solubility and chemical
stability
Recovery of MTZS and MTZ in feces and urine after
oral administration of drugs
e apparent partition coefficients in 1-octanol/water
system for MTZ and MTZS were 0.95 and 0.15, respec-
tively. While the solubility of MTZ was 10.5 mg/mL,
MTZS was freely soluble. To examine the chemical stabil-
ity of MTZS during the transit through the GI tract, MTZS
was incubated in pH 1.2, 4.5 and pH 7.4 buffer solutions.
Degradation of MTZS and generation of MTZ was negli-
gible up to 10 h.
Male SD rats (250–260 g) were placed in a metabolic cage
and starved for 24 h prior to use for the experiments but
had free access to water. MTZ or MTZS (4 mg equivalent
of MTZ) was orally administered. e fecal and urinary
samples were collected separately at a 2-h interval and
stored immediately in a freezing cabinet at −20°C until
analysis. e fecal or urinary samples were 10-fold
diluted with 0.05 M pH 6.8 isotonic phosphate buffer
solution (Kim et al., 2009). e samples were vortexed
and centrifuged at 5000 rpm for 3 min. To a 100 µL por-
tion of the supernatant, was added 900 µL of methanol to
precipitate protein in the samples, vortexed for 2 min, and
centrifuged for 5 min at 14,000 rpm. e concentration of
MTZ and/or MTZS in a 20 µL portion of the supernatant
was analyzed by HPLC.
Incubation of MTZS in the contents of various
segments of the rat GI tract
We examined whether MTZS could be delivered to the
large intestine without significant loss in the upper intes-
tine and liberate MTZ at the target site. MTZS (100ppm
equivalent of MTZ) was incubated with the contents
of various segments of the rat GI tract monitoring the
change of the MTZS level. As shown in Figure 2A, after
10 h incubation, the level of MTZS decreased to 82, 90 and
10% of the dose in the contents of PSI, DSI and cecum,
respectively, indicating that, while relatively stable in
the contents of the upper intestine, MTZS is degraded
rapidly in the cecal contents. To examine the effect of the
concentration of the cecal contents on the disappearance
of MTZS, MTZS was incubated in the cecal contents at
various concentrations. As shown in Figure 2B, MTZS
disappeared during the incubation, which was acceler-
ated as the concentration of the cecal contents increased.
Although the sulfate conjugate was degraded extensively,
MTZ was not detected in the cecal contents. We hypoth-
esized that MTZ liberated from MTZS also metabolizes
in the cecal contents, the rate of which is greater than
that of the liberation of MTZ from MTZS, resulting in
no accumulation of MTZ. To test this hypothesis, MTZ
was incubated in the cecal contents at various concen-
trations and the rates of disappearance were compared
with those of MTZS. As shown in Figure 2C, consistent
with our hypothesis, MTZ disappeared much faster
than did MTZS in the cecal contents. To see MTZ liber-
ated from MTZS in the cecal contents, MTZS at elevated
concentrations (250 and 500 ppm) was incubated in 5%
cecal contents. While hardly detected on incubation of a
250 ppm concentration of MTZS (data not shown), MTZ
was detected at a maximal level of 3.3 ppm on incubation
of a 500 ppm concentration of MTZS (Figure 2D).
Statistical analysis
e results are expressed as the mean SE. One-way
ANOVA followed by Tukey’s (HSD) test was used for test-
ing the difference between data. Differences with p< 0.05
were considered significant. e XLSTAT® Software
(Addinsoft, Inc, Suite 503, NY) was used for the statistical
analysis.
Results
Preparation of MTZS
MTZS was prepared by the synthetic method as shown in
Figure 1. Sulfate was easily conjugated with the hydroxyl
Figure 1. Synthesis of metronidazole sulfate. MTZS was prepared
as described under “Material and Methods”. MTZ, metronidazole;
MTZS, sulfate conjugated metronidazole.
Journal of Drug Targeting

A colon-specific prodrug of metronidazole 259
Figure 2. Stability of MTZS in the contents of various segments of the GI tract of rats. (A) Metronidazole sulfate (MTZS, 100ppm equivalent
of MTZ) was incubated with the 5% contents of various segments of the GI tract of rats. e concentration of MTZS was determined at
appropriate time intervals. (B) MTZS (100ppm equivalent of MTZ) was incubated with the cecal contents at various concentrations.
e concentration MTZS was determined at appropriate time intervals. (C) Metronidazole (MTZ, 100ppm) was incubated with varied
concentrations of the cecal contents of rats. e concentration of MTZ was determined at appropriate time intervals. (D) Metronidazole
sulfate (MTZS, 500ppm) was incubated with 5% cecal contents of rats. e concentrations of MTZ and MTZS were determined at appropriate
time intervals. (E) MTZ (100ppm) or MTZS (100ppm equivalent of MTZ) was incubated in 5% cecal contents which was autoclaved at
121°C for 20min. e concentration of either MTZ or MTZS was determined at appropriate time intervals. e data in “A”, “B”, “C”, “D” and
“E” are mean SE (n=5).
We examined whether the disappearance of MTZS
and MTZ was dependent on microbial enzymes. e
cecal contents was autoclaved to inactivate microbial
enzymes (Kim et al., 2005) followed by addition of MTZS
or MTZ and the change in their levels was monitored. No
significant change was detected up to 10 h (Figure 2E).
Since amoebic infection causes colitis, and MTZ is also
used for the treatment of inflammatory bowel disease, we
examined whether inflammation affected the metabo-
lism of MTZS and MTZ. To do this, the metabolic change
of MTZS and MTZ was monitored in the cecal contents at
various concentrations obtained from normal rats or rats
with TNBS-induced colitis. As shown in Figure 3A and
3B, no significant difference was observed.
examined whether the disappearance of MTZ in the cecal
contents was relevant to the metabolism to the active
metabolite. We first tested whether MTZ metabolized
by microbes in the cecal contents. To do this, the cecal
contents were centrifuged followed by filtration of the
supernatant using a filter membrane with 0.22-µm pore
size and MTZ was incubated in the filtered supernatant.
No change of the MTZ level was observed up to 10 h. To
further examine the relevance of the disappearance of
MTZ with the bioactivation of MTZ, MTZ was incubated
with the cecal contents pretreated or left untreated with
dicumarol, a nitroreductase inhibitor (Kinouchi and
Ohnishi, 1983), and the change of MTZ level was moni-
tored. As shown in Figure 4A, the reductase inhibitor
delayed the disappearance of MTZ. To experimentally
prove that the cecal metabolisms of MTZ and MTZS were
associated with antimicrobial activity, MTZS or MTZ
was treated in the cecal contents diluted with LB broth
where a dialysis tube containing E. Coli was immersed
and antimicrobial activity of the drugs was determined
Cecal metabolism of MTZ is relevant to the
bioactivation for antimicrobial action
Since MTZ enters protozoans or bacteria and the nitro
group is reduced to give an amoebicidal and bactericidal
metabolite (Ludlum et al., 1988; Lamp et al., 1999), it was
© 2012 Informa UK, Ltd.

260 H. Kim et al.
Figure 3. e effect of colonic inflammation on the cecal metabolism of MTZ and MTZS. (A) Metronidazole (MTZ, 100ppm) was incubated
with varied concentrations of the cecal contents obtained from normal or colitic rats. e concentration of MTZ was determined at
appropriate time intervals. (B) e same experiment was done with MTZS (100ppm equivalent of MTZ). e data in “A” and “B” are mean
SE (n=5).
Figure 4. e cecal metabolism of MTZ is relevant to bioactivation of MTZ. (A) Metronidazole (MTZ, 200ppm) was incubated with the
10% cecal contents of rats in the presence of dicuomarol (Dcm). e concentration of MTZ was determined at appropriate time intervals as
described under. e data are mean SE (n=5). (B) e cecal contents were diluted with LB broth to afford the 20% (w/v) suspension. 5 mL
portion of MTZ or MTZS solution (equivalent to 500 ppm MTZ) in LB broth was mixed with 5 mL of the cecal contents immediately followed
by immersion of a dialysis tube in each cecal suspension, which was incubated at 37°C in a shaking incubator in a nitrogen glove bag. For
comparison, the same experiment was done without the cecal contents. e antimicrobial activity of MTZ and its prodrug was determined
by measurement of O.D. of the E. Coli 8h after incubation. Control (C): group untreated with either MTZS or MTZ (*p<0.001 vs. control;
**p>0.05 vs. control; ***p<0.005 vs. MTZ; #p<0.01 vs. control; ##p<0.05 vs. control; ###p>0.05 vs. MTZ).
by measurement of O.D. of the E. Coli, which is one of
methods to evaluate bacterial growth, 8 h after the incu-
bation. For comparison, the same experiment was done
without the cecal contents. As shown in Figure 4B, MTZS
exhibited an antibacterial activity as potent as MTZ in the
cecal contents whereas the antibacterial activity of MTZS
was negligible compared to that of MTZ in the LB broth
(without the cecal contents).is result strongly suggests
that MTZS is microbially converted to MTZ, which is
subsequently bioactivated in colonic microbes to exhibit
antibacterial activity.
the same experiment was done with MTZ. As shown in
Figure 5A, while oral administration of MTZ gave a sig-
nificant amount of the drug in the blood (Cmax: 7.8 µg/mL,
T
max: 1.6 h), no MTZS in the plasma was detected through-
out the whole experimental period after oral administra-
tion of MTZS. To further confirm the limited systemic
absorption of MTZS, the concentration of MTZS in the
urine collected for 24 h was determined after oral admin-
istration of MTZS. For comparison, the same experiment
was done with MTZ. As shown in Figure 5B, the urinary
recovery of MTZS was about 3.3% of the dose after oral
administration of MTZS. After oral administration of
MTZ, about 11.2% of the dose was excreted as an intact
form and a small amount of metronidazole sulfate, one
of the liver metabolites of MTZ (Loft and Poulsen, 1989),
was observed in the urine. To exclude the possibility that
the low urinary recovery of MTZS is due to metabolic
changes of MTZS in the blood and/or the liver, MTZS was
Systemic absorption of orally administered MTZS
Although the low apparent partition coefficient of MTZS
suggests limited systemic absorption upon oral adminis-
tration of the prodrug, we wanted to examine the notion
in vivo. MTZS was administered orally and plasma con-
centration of MTZS was determined. For comparison,
Journal of Drug Targeting

A colon-specific prodrug of metronidazole 261
Figure 5. e systemic absorption of orally administered MTZS. (A) MTZS (4mg equivalent of MTZ) was administered orally to rats (250–
260g) and the plasma concentration of MTZS was determined at appropriate time intervals. For comparison, the same experiment was
done with MTZ (4mg). (B) e same experiment was done with MTZS (4mg equivalent of MTZ) as described in “A” except for monitoring
MTZS level in the urine. (C) MTZS (100ppm equivalent of MTZ) was incubated in the 10% liver homogenates of rats. e concentration of
MTZS was determined at appropriate time intervals. e same experiment was done with MTZ (100 ppm). (D) MTZS (100ppm equivalent
of MTZ) was incubated in the 80% plasma of rats. e concentration of MTZS was determined at appropriate time intervals. e same
experiment was done with MTZ (100ppm). e data in “A”, “B”, “C” and “D” are mean SE (n=5).
Figure 6. Distribution of MTZ and MTZS in the GI tract and fecal recovery after oral administration of MTZS. (A) MTZS (4 mg equivalent of
MTZ) was administered orally to rats (250–260g). e concentration of MTZS and MTZ was determined in the lumens of the proximal small
intestine (PSI), distal small intestine (DSI), cecum and colon of rats at appropriate time intervals. For comparison, the same experiment
was done with MTZ (4mg). (B) MTZS (4mg equivalent of MTZ) was administered orally to rats (250–260g). e fecal samples were collected
separately at a 2-h interval. e concentration of MTZS and MTZ was analyzed. For comparison, the same experiment was done with MTZ
(4mg). e data in “A” and “B” are mean SE (n=5).
incubated with the 80% plasma and the 10% liver homo-
genates. For comparison, the same experiment was done
with MTZ. While MTZ metabolized rapidly in the liver
homogenates, the level of MTZS was not changed up to
for 12 h (Figure 5C). e levels of MTZS and MTZ were
not changed in the plasma (Figure 5D).
administered MTZS can reach the large intestine fol-
lowed by liberation of MTZ at the target site. To verify this
in vivo, MTZS was administered orally to rats and MTZS
and MTZ were monitored in PSI, DSI, cecum, colon and
feces at various time intervals. For comparison, the same
experiment was done with MTZ. While MTZ was not
detected anywhere in the GI tract 1h after oral adminis-
tration of MTZ (data not shown), as shown in Figure 6A,
MTZS,whichpassedthePSI,wasaccumulateduptoabout
0.7 mM (175 µg/mL) in the DSI and then moving down to
the large intestine. While MTZS was detected very low in
Colonic delivery and systemic absorption of MTZ
following oral administration of MTZS
e results of in vitro incubation and the systemic
absorption of MTZS suggest that a large fraction of orally
© 2012 Informa UK, Ltd.

262 H. Kim et al.
the colon, the maximal concentration of MTZS reached
about 0.28 mM (70 µg/mL) at the cecum 3 h after admin-
istration. Moreover, after oral administration of MTZS,
MTZ was not detected at all in the upper intestine and
hardly detected in the large intestine probably resulting
from rapid colonic metabolism of MTZ liberated from
MTZS (data not shown). On the other hand, as shown in
Figure 6B, MTZS was not observed and a little amount of
MTZ (15 µg) was recovered in the feces collected for 24 h
after administration of MTZS. e fecal recovery was 0.4%
of the dose. On the contrary, either MTZ or MTZS was not
detected at all in the feces after administration of MTZ
(Figure 6B). e systemic absorption of MTZ is related to
its adverse effects. We examined whether the prodrug of
metronidazole was able to reduce the systemic absorp-
tion of MTZ in addition to the efficient colonic delivery
of MTZ. MTZS was administered orally to rats and the
plasma concentration and urinary excretion of MTZ was
monitored. MTZ was not detected in the plasma and
urine throughout the whole experimental period (24h)
upon oral administration of MTZS (data not shown).
upper intestine; (ii) MTZ was not at all detected in the
upper intestine although a large amount of MTZS was
monitored after oral administration of MTZS. It would
be natural that the level of MTZS in the large intestine
be lower than that of MTZS in the DSI, considering rapid
metabolism of MTZS in the cecal contents.
Although MTZ was not detected in the large intestine
after oral administration of MTZS, it is very likely that
MTZS delivered to large intestine is converted to MTZ and
subsequently exerts an anti-protozoal action. is specula-
tion is rationalized by the data demonstrating that (i) MTZ
also metabolized as quickly as did MTZS in the cecal con-
tents, (ii) dicumarol, a nitroreductase inhibitor, delayed
the disappearance of MTZ during incubation in the cecal
contents and nitroreduction of MTZ is a microbial metab-
olism that generates an active metabolite to kill microbes
(Ludlum et al., 1988; Lamp et al., 1999), (iii) MTZS elicited
antibacterial activity in the cecal contents, which was as
potent as MTZ. is argument was further supported by
showing that MTZS at an elevated concentration (500 µg/
mL) disappeared as MTZ appeared although MTZ was
produced at much lower amount than MTZS disappeared.
Our data showing that MTZ did not at all disappear when
incubated in the microbe-free cecal contents and the
autoclaved cecal contents imply that such a microbial
metabolism occurred to MTZ in the cecum.
Since our data showed that induction of inflamma-
tion, which reportedly affects the metabolism of drugs
in the large intestine (Marteau et al., 2003), made no big
difference in the metabolisms of MTZ and MTZS, MTZS
is likely to exhibit similar therapeutic efficiency regard-
less of inflammation in the large intestine. In addition to
a greater delivery of MTZ to the target site, MTZS should
reduce adverse effects by systemic absorption of MTZ.
We demonstrated that MTZ was not detected in the blood
and the urine after oral administration of MTZS while
MTZ administered orally gave a significant amount of
MTZ in the blood and the urine. Taken together, our data
suggest that MTZS is a promising colon-specific prodrug
of MTZ which could improve therapeutic and toxicologi-
cal properties.
Jung et al. has suggested that to have various colon-
specific promoieties coupling with a functional group
is one strategy to overcome obstacles in developing a
practical colon-specific prodrug such as inter and intra-
individual variation in prodrug activation and the colonic
metabolism of parent drugs (Jung and Kim, 2010). e
conversion of the colon-specific prodrugs (with different
promoieties) to the drugs is carried out by different acti-
vation systems (bacterial enzymes) in the large intestine,
which may provide an option minimizing variation in the
prodrug activation. In addition, controlling the colonic
distribution of the active parent drug liberated from a
colon specific prodrug is also important for treatment of
colonic diseases in which a pathological lesion may be
present at various regions. In case, a parent drug is very
susceptible to the colonic metabolism (as MTZ), the
parent drug converted from its colon-specific prodrug
Discussion
In this study, we in vitro/in vivo-evaluated MTZS as a
potential colon-specific prodrug of MTZ. MTZS was
(bio)chemically stable and non-absorbable in the upper
intestine resulting in the efficient colonic delivery of
MTZ. Furthermore, MTZS was converted to MTZ in the
large intestine followed by rapid disappearance of MTZ
probably resulting from the metabolism to an active
metabolite. In addition to the efficient colonic delivery
of MTZ, MTZS reduced the systemic absorption of MTZ,
which is related to its adverse effects.
In our previous papers, sulfate conjugated steroids
were developed as a colon-specific prodrug of the ste-
roids (Jung et al., 2003; Kim et al., 2006). We applied the
delivery system to MTZ, a drug of choice for luminal
amoebiasis. As expected, the hydroxyl group in MTZ was
readily sulfated to afford MTZS. e delivery system con-
ferred a greater hydrophilicity to MTZ as indicated by the
lowered apparent partition coefficient, suggesting that
systemic absorption of MTZS would be limited. is was
verified by the in vivo data demonstrating that MTZS was
not detected in the blood and was recovered at less than
5% of the dose in the urine after oral administration of
MTZS. Since MTZS was stable in the plasma and the liver
homogenate, it would be reasonable to assume that the
MTZS in the urine mainly comes from MTZS absorbed
systemically from the upper intestine. MTZS was likely
to be undetectable in the blood because of low systemic
absorption and rapid excretion of MTZS, an anionic sul-
fate prodrug.
Most of the prodrug, which is not absorbed in the
upper intestine, seems to be delivered to the large intes-
tine. is argument is supported by the in vitro and
in vivo data showing that (i) MTZS is stable not only in
pH 1.2 and pH 7.4 buffers but also in the contents of the
Journal of Drug Targeting

A colon-specific prodrug of metronidazole 263
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should be metabolized rapidly during moving down the
colon and could not reach a pathological lesion distant
from the conversion site. For this reason, the colonic dis-
tribution of the drug would depend mainly on the con-
version rate of its prodrug in the large intestine, which
may vary according to promoieties (Jung and Kim, 2010),
erefore, along with N-nicotinoyl-(2-acetoxy)-D,L-
glycine that is previously reported as a colon-specific pro-
moiety of MTZ (Kim et al., 2009), sulfate ester promoiety
should make a contribution to development of a clinically
useful colon-specific prodrug of MTZ. Moreover, the syn-
thetic process of the sulfation is simple and usually gives
high yield compared with those of other delivery systems
such as azo bond, glycoconjugation, dextran conjuga-
tion, N-nicotinoyl-(2-acetoxy)-D,L-glycine and amino
acid conjugation (Friend and Chang, 1985; McLeod et al.,
1994; Jung et al., 2000; Lee et al., 2001; Jain et al., 2006).
is may be of great advantage for industrialization of a
colon-specific prodrug.
Declaration of interest
is work was supported by the Korea Research
Foundation (KRF) grant funded by the Korea govern-
ment (MEST) (KRF-2008-314-E00315 and KRF-2007-
314-E00245).
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