Zopolrestat (Alond) is a new drug that is being evaluated as an
aldose reductase inhibitor for the treatment of diabetic complications. 14C-labeled zopolrestat was orally administered
to rats for a tissue distribution study and a bile duct cannulation
metabolism study. Tissue samples from the distribution study were
analyzed by complete oxidation and liquid scintillation counting. Urine
and bile samples from the bile duct cannulation study were analyzed by
microbore HPLC, with simultaneous radioactivity monitoring and
atmospheric pressure ionization tandem mass spectrometry. The mass
balance in the distribution study demonstrated that the greatest
exposure (AUC0-
) occurred in the liver,
followed by the ileum and large intestine. The time of maximal plasma
concentrations for nearly all tissues was 4 hr after the dose, and the
half-life of radioactivity in most tissues (8-10 hr) was similar to
the half-life in plasma. For the bile duct-cannulated rat study, most of the radioactivity was recovered in the bile, indicating that biliary
excretion is a major route of elimination of zopolrestat and its
metabolites in rats. Numerous oxidative metabolites, as well as phase
II conjugates, were identified in the bile and urine samples. Acyl
glucuronides of zopolrestat and unchanged drug accounted for >85% of
biliary radioactivity, whereas unchanged drug and degradation products
of glutathione conjugates were identified as the major urinary metabolites.
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Introduction |
Methods
for the treatment and prevention of the long-term
complications of diabetes are medically needed. In the past 10 years, it has been shown that flux through the polyol pathway in diabetic animals and humans may be associated with complications such as neuroppathy, nephropathy, cataractogenesis, and macrovascular disease (Kinoshita and Nishimura, 1988
; Nathan, 1995; Raskin and Rosenstock, 1987; Sastry et al., 1995). The enzyme aldose
reductase has been implicated in the pathogenesis of diabetic
complications such as these (Lee et al., 1995; Sastry
et al., 1995). The use of aldose reductase inhibitors in
diabetic subjects has been shown to have a positive impact on motor
nerve conduction velocity, sciatic nerve blood flow, tissue sorbitol
levels, and sciatic nerve fructose and myo-inositol levels
(Beyer-Mears et al., 1984; Gonzalez et
al., 1983; Hotta et al., 1995; Whiting and Ross, 1988). Several aldose reductase inhibitors have been shown to decrease erythrocyte sorbitol levels in human subjects (Malone et
al., 1984; Raskin et al., 1985).
Zopolrestat (CP-73,850)
[3,4-dihydro-4-oxo-3[[5-(trifluoromethyl)-2-benzothiazolyl]methyl]-1-phthalazineacetic
acid] is a novel, carboxylic acid, aldose reductase inhibitor. It has
been shown to normalize sorbitol, fructose, and myo-inositol
levels in sciatic nerve, lens, retina, and kidney of diabetic rats
(Beyer et al., 1990; Mylari et al., 1991) and to
normalize renal plasma flow in galactosemic rats (Oates and Ellery,
1990). The pharmacokinetics of zopolrestat have been evaluated in
normal and diabetic rats (Inskeep et al., 1991), healthy
male volunteers (Inskeep et al., 1994), and
non-insulin-dependent diabetic patients (Inskeep et al.,
1995).
The pharmacological and pharmacodynamic effects of zopolrestat are
briefly summarized above, but details of the metabolism and disposition
of zopolrestat have not yet been reported. To assess the metabolism and
disposition of zopolrestat in rats after a radioactive dose, we used
simultaneous radioactivity detection and atmospheric pressure
ionization MS with an electrospray ionization interface. Atmospheric
pressure ionization MS has evolved into a common analytical tool that
provides a soft ionization technique for many analytes. It has been
extensively used for the quantitative analysis of drugs (Fouda and
Schneider, 1995), the identification of neurotoxic metabolites
(Subramanyam et al., 1991), the confirmatory identification
of drug residues (Schneider et al., 1993), and the
elucidation of the metabolic fate of drugs (Tomlinson et
al., 1993). The studies described here made extensive use of these methods, and they summarize the metabolic profile of zopolrestat in
rats after oral administration of radiolabeled drug.
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Materials and Methods |
Labeled Zopolrestat.
Radiolabeled zopolrestat (fig. 1)
was synthesized at Pfizer Central Research (Groton, CT) (Mylari and
Zembrowski, 1991). The specific activity of
[14C]zopolrestat (lot 22280-236-2, C-725) used
in this study was 1.05 µCi/mg.
Reagents.
All solvents and reagents were of HPLC grade and were purchased from
Fisher Scientific (Fair Lawn, NJ). Ecolite scintillation cocktail was
purchased from ICN (Irvine, CA). Permafluor E+ scintillation fluid,
Ultima Gold liquid scintillation fluid, and Carbo-Sorb trapping solvent
were purchased from Packard Instrument Co. (Meriden, CT). Putative
metabolites (CP-72,262, 5-trifluoromethyl-2-benzothiazol; CP-114,914,
phthalazineacetic acid) were synthesized at Pfizer and used as
standards for HPLC and MS reference.
Animal Studies. Tissue Distribution Study.
Eighteen male Long-Evans rats (body weight, 315 ± 9 g) were
dosed with an aqueous solution of
[14C]zopolrestat (5.10 mg/ml; specific
activity, 0.92 µCi/mg) by gavage, for a total dose of 57 ± 2 mg/kg. Rats were randomly assigned to one of six groups (three
rats/group) and sequentially numbered. Groups were sacrificed at 4, 8, 24, 48, 96, or 168 hr after dosing, and eyes, brain, lungs, heart,
pancreas, spleen, adrenal glands, kidneys, testes, light pigmented
skin, dark pigmented skin, sciatic nerves, muscle tissue (from the
thigh), urinary bladder, blood, stomach, liver, jejunum, ileum, large
intestine, and abdominal fat were dissected and sampled. Stomach and
intestinal tissues were separated from contents, and the tissues were
rinsed with water. In most cases, three portions (approximately
0.1-0.4 g) of each tissue were transferred to tared combustion cups
and weighed. Eyes and sciatic nerves were collected as duplicate
samples, and adrenal glands and urinary bladder (contents removed) were
analyzed as single samples.
Tissue levels of radioactivity were expressed as
microgram-equivalents per gram (i.e. dpm per gram divided by
the specific activity of the parent drug) and represented the mean of
replicate determinations, except for adrenal glands and urinary bladder (single determinations were made because of the small amount of tissue
available). The lower limit of quantification for tissue radioactivity
levels depended on the amount of tissue mass available for sample
oxidation. Samples were counted for up to 30 min, with minimal
quantifiable counts (generally in the range of 80-120 dpm) being
defined as approximately 3 times background counts. The approximate
lower limits of quantification (and approximate tissue masses) were 0.4 µg-eq/g (0.1 g) for eyes, 0.8 µg-eq/g (0.05 g) for adrenal glands,
1.5 µg-eq/g (0.04 g) for sciatic nerve, 0.4 µg-eq/g (0.1 g) for
whole blood, plasma, and red blood cells, 0.3 µg-eq/g (0.15 g)
for fat, and 0.15 µg-eq/g (0.3 g) for all other tissues.
Pharmacokinetic parameters were calculated from mean tissue levels.
AUC0-t was calculated using a linear
trapezoidal approximation. The elimination rate constant was determined
from the slope of the regression line of ln (tissue concentration) vs. time. AUC0- was calculated by
adding to AUC0-t the mean terminal tissue
concentration divided by the elimination rate constant.
Bile Duct-Cannulated Rat Study.
The bile ducts of several male or female Long-Evans rats were
cannulated so that continuous collections of bile could be obtained for
at least 48 hr from four male and four female rats. Data from additional animals that were dosed for use as substitutes for animals
that did not survive surgery or did not provide bile for at least 48 hr
were not included in the analysis. Male rats were dosed with
approximately 12 ml of dosing solution/kg of body weight, with a 8.02 mg/ml solution of [14C]zopolrestat, and female
rats were dosed with approximately 20 ml of dosing solution/kg of body
weight, with a 4.63 mg/ml solution. The total dose was approximately
100 mg/kg. The rats were housed in metabolism cages. Urine, bile, and
feces samples were collected for 0-24 hr on days 1, 2, 3, and 4. Urine
collection vessels contained 0.5 ml of 1.0 M phosphoric acid for male
rats and 0.5-1.0 ml of 2 M phosphoric acid for female rats. All
samples were stored frozen and were thawed just before assay.
Total Radioactivity Determination. Tissue Distribution
Study.
Radioactivity in all tissue samples was determined by sample oxidation
in a Packard oxidizer (model 306). Burn efficiency standards were used
throughout all sample oxidizer runs. Samples were counted for up to 30 min in a Packard scintillation counter (model 2000CA), and counting
efficiency was determined using blank oxidizer samples (processed
through a burn cycle) fortified with [14C]toluene (18,330 dpm). For some samples,
counting efficiency was determined by adding internal standard
([14C]toluene) to the sample and recounting.
Bile Duct-Cannulated Rat Study.
Radioactivity in bile and urine was determined by
LSA1 after dilution in
Packard Ultima Gold liquid scintillation fluid. Radioactivity in feces
was determined by LSA after sample oxidation in a Packard sample
oxidizer, using Packard Carbo-Sorb trapping solvent. The oxidized
samples were diluted with Packard Permafluor E+ scintillation fluid.
Internal standardization ([14C]toluene) was
used for all LSA determinations in this study.
Quantitative Profiling of Radioactivity for Cannulated Rat Study
Samples. Urine Profiling.
Urinary metabolite profiles were determined for each animal, using
individual or pooled urine samples containing at least 70% of total
urinary radioactivity. When urine samples from one animal were
combined, aliquots from each interval were combined in the same
proportion as the total urine volumes for the intervals. In one case
(male 20), profiles were determined in two separate urine samples, and
the results for the composite sample were calculated with a similar
volume-weighted summation.
Aliquots (0.5 ml) of thawed urine samples were centrifuged, and the
supernatant was transferred to another tube. The remaining pellet was
quantitatively extracted with 1 ml of ACN. The ACN extract was
evaporated to dryness, and the 0.5-ml urine supernatant layer was added
to the residue. The samples were flash-frozen and lyophilized. The
residues of the urine samples were reconstituted in 60 µl of 20 mM
ammonium acetate plus 40 µl of ACN. Aliquots (10 µl) were injected
into a microbore HPLC system, and two-drop (approximately 30-sec)
fractions were collected. Additional 10-µl aliquots of reconstituted
samples were removed to determine total radioactivity injected onto the
HPLC column. The amount of radioactivity in each fraction and in the
10-µl recovery aliquot was determined by LSA after addition of 6 ml
of Ultima Gold scintillation fluid. Metabolite peaks were quantified
based on retention times, relative to that of unchanged drug. Each
metabolite was quantified by the amount of radioactivity in the
fractions collected at its HPLC retention time.
Bile Profiling.
To quantify biliary radioactivity, 5-µl injections of bile
(containing ~3000 dpm) were made. Bile samples that exhibited low activity were concentrated ~2-3-fold by an ACN precipitation method. With this procedure, mean recoveries of radioactivity were 89%.
A 
-RAM (IN/US Systems, Tampa, FL) was equipped with a specially
designed cell to accommodate low flow rates (i.e. 75 µl/min). The flow cell minimized extracolumn effects during
radioactivity monitoring, with reduced tubing inner diameter and volume
(cell tubing i.d., 0.028 inch; total cell volume, 100 µl). A Harvard syringe pump with a 10- or 25-ml syringe was used to deliver the cocktail to the liquid scintillation flow cell. The connecting tubing
between the MS ion-spray interface and the -RAM was a combination of
50-µm i.d. fused silica (to the ion-spray interface) and 100-µm
i.d. fused silica (to the -RAM). With this configuration, the LC
flow rate of 75 µl/min was split, such that the mass spectrometer received ~15 µl/min and the -RAM received ~60 µl/min.
After chromatographic resolution, the -RAM was used to quantify the
radioactivity associated with each biliary metabolite. The -RAM
provided peak representation, an integrated printout of dpm/peak, and
the percentage of radiolabeled material in each peak, relative to the
total amount of activity detected. The -RAM was operated in the
homogeneous liquid scintillation counting mode, with the addition of
scintillation cocktail to the LC effluent at 240 µl/min. Total flow
to the -RAM was ~300 µl/min, resulting in a cell residence time
of 20 sec.
Recovery of biliary radioactivity from the HPLC column was determined
in a separate experiment. After injection of 5 µl of bile, the HPLC
effluent was collected and counted by LSA. The recovery of
radioactivity was approximately 96%.
HPLC. Urine Analysis.
HPLC analysis used a Keystone BDS
Hypersil C18 column (1 × 250 mm). An ABI
140A dual-syringe pump provided a constant flow of 75 µl/min. The
mobile phase and linear gradient conditions were as follows: solvent A,
1:9 ACN/20 mM NH4OAc (pH 5); solvent B, 9:1
ACN/20 mM NH4OAc (pH 5); at 0 min, A = 100%; at 15 min, A = 80%; at 22 min, A = 50%; at 30 min,
A = 50%; at 33 min, A = 90%; at 41 min, A = 100%. The
column was equilibrated for an additional 15 min before the next
injection. Under these conditions, zopolrestat eluted at approximately
24 min.
Bile Analysis.
The analytical column and syringe pump were the same as described
above. The mobile phase and linear gradient conditions were as follows:
solvent A, 1:9 ACN/20 mM NH4OAc (pH 4); solvent
B, 9:1 ACN/20 mM NH4OAc (pH 4); at 0 min, A = 90%; at 30 min, A = 70%; at 35 min, A = 35%; at 45 min,
A = 35%; at 50 min, A = 90%. The column was equilibrated
for an additional 15 min before the next injection. Under these
conditions, zopolrestat eluted at approximately 35 min.
Preparation of Biological Samples for MS Analysis.
For the characterization of urinary metabolites, aliquots of pooled
male rat urine (1 ml; 0-24 hr after the dose) containing 392,000 dpm/ml were acidified with approximately five drops of 2 N HCl and
vortex-mixed. Samples were extracted with 5 ml of diethyl ether by
shaking for 20 min, followed by centrifugation. The ether was
transferred to a clean tube and vortex-evaporated (40°C) for 15 min.
The residues were reconstituted in 0.5 ml of ACN, vortex-mixed, and
washed with 2-3 ml of n-hexanes. After centrifugation, the
hexane was aspirated to waste, and the ACN was vortex-evaporated to
dryness (approximately 25 min at 40°C). The residues were
reconstituted in 25 µl of ACN, vortex-mixed, and sonicated, and 75 µl of 20 mM NH4OAc (adjusted to pH 5 with acetic acid) was added. After a final vortex-mixing, injections of 5 µl were made into the HPLC/-RAM/MS system. The extraction efficiency of this procedure was determined in a separate experiment. Liquid scintillation radioactivity measurements indicated that approximately 76% of the radioactivity in urine was recovered.
To characterize biliary metabolites, a 50-µl aliquot of rat bile was
diluted with an equal volume of HPLC mobile phase (2:8 ACN/20 mM
NH4OAc, pH 4). After vortex-mixing, injections of
5 µl were made into the HPLC/-RAM/MS system.
Simultaneous MS and -RAM Detection.
A Sciex API III+ biomolecular mass analyzer was used for metabolite
identification. The ion-spray interface was operated in the positive-
or negative-ion mode, at a voltage of ±5000 V. The atmospheric
pressure chemical ionization interface was operated in the
positive- or negative-ion mode, and a corona discharge current of ±4
µA was established. CID studies were performed with argon as the
collision gas and with collision gas thickness values of 2.0-2.4 × 1014 atoms/cm2.
Collision energies were typically 
23 eV. Metabolites were identified by simultaneous -RAM detection and corroborating MS analysis. MS
experiments included Q1 and product-ion scanning of relevant ions.
Other informative MS/MS experiments included the following: precursor-ion scanning of m/z 204, indicative of a
benzothiazole moiety; precursor-ion scanning of m/z 390, indicative of an oxidized m/z 374 product ion; precursor-ion
scanning of m/z 452, indicative of a thiol addition;
neutral-loss scanning of 129 amu, indicative of mercapturic acid conjugates.
The HPLC column effluent was split before the ion-spray needle. The
-RAM response was also recorded by the mass spectrometer, providing
simultaneous detection of radioactivity and MS data. The delay in
response between the two detectors was approximately 0.28 min, with the
MS data being recorded first.
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Results |
Tissue Distribution Study:
Maximal levels of radioactivity from
[14C]zopolrestat were observed at 4 hr after
dosing in all tissues except urinary bladder and large intestine, where
maximal levels were observed at 8 hr after dosing (table
1). Radioactivity could be quantified in all tissues, except eyes, brain, and adrenal glands, for 48 hr after dosing. Only in testes and liver could radioactivity still be quantified 168 hr after dosing, and levels in these tissues were
very close to the lower limits of quantification (approximately 0.15 µg-eq/g).
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TABLE 1
Pharmacokinetics of radioactivity from [14C]zopolrestat in
male rats after a single oral dose of 57 mg/kg
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Tissue exposure to radioactivity, as expressed by
AUC0-, was greatest in liver, followed by the
intestines, kidney, and plasma (table 1). Exposure to radioactivity for
all other tissues was 3-30-fold lower than for liver.
Tissue radioactivity half-life was longest in testes (46 hr).
Half-lives in skin, sciatic nerve, and liver were approximately 20 hr,
and half-lives in all other tissues were <13 hr (table 1).
Bile Duct-Cannulated Rat Study. Total Radioactivity Excreted
in Urine, Bile, and Feces.
Most of the radioactivity administered to the rats in this study was
recovered in urine, bile, and feces within 2 days after dosing. The
bile contained the most radioactivity (59.70-73.67% of the dose for
individual males and 12.53-52.10% for individual females). Means ± SD of recoveries of administered radioactivity for the four male
rats during the 4 days after dosing were 12.30 ± 4.59% in urine,
66.24 ± 6.45% in bile, and 14.83 ± 3.72% in feces (table
2). Means ± SD of recoveries of
administered radioactivity for the four female rats were 19.11 ± 6.43% in urine, 32.25 ± 19.37% in bile, and 11.90 ± 7.07% in feces. Small amounts of radioactivity (<3% of the dose)
were recovered in cage washes and in residual food. Mean total recovery
of the administered dose was 94.20 ± 6.90% for male rats and
65.00 ± 14.22% for female rats. The recoveries of administered
radioactivity in female rats might have been lower because the bile
duct cannulations remained patent for 4 days for three of four males
but for only one of four females. Patency for two of the females lasted
for only 2 days. Total recovery of radioactivity in the two females
with better patency was similar to total recovery in the four males.
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TABLE 2
Recoveries of radioactivity from Long-Evans rats after oral
administration of [14C]zopolrestat at 100 mg/kg
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MS Analysis of Zopolrestat and Identification of Metabolites.
A CID spectrum of zopolrestat is shown in fig.
2. Structural assignments for the major
product ions are noted. The structures of the proposed urinary and
biliary metabolites are shown in fig. 3,
and the structural descriptions are listed in table
3. Each metabolite was assigned a
numerical notation (metabolite 1, 2, etc.) based on the order of HPLC
elution.
Identification of Urinary Metabolites.
Female rats excreted more radioactivity into urine than did male
rats (table 2), and most of the difference was attributable to
unchanged drug. Whereas males excreted 1.3 ± 1.3% of the dose in
urine as unchanged drug in 24 hr, females excreted 6.9 ± 3.5% of
the dose in 24 hr, plus an additional 9.5 ± 4.4% of the dose between 24 and 48 hr. Radioactivity from urine samples injected into
the HPLC system was quantitatively recovered in the column fractions.
In male rats, most of the radioactivity in urine was attributable to
metabolites 8 and 9 (table 4 and fig.
4). Parent drug was the second most
abundant form of radioactivity in the urine of males. All other
identified metabolites (metabolites 6 and 7, 10 and 11, and 12) each
accounted for approximately 10% of total urinary radioactivity. In
female rat urine, the same metabolites were observed at approximately
the same concentrations as in male urine. However, because of the high
concentrations of parent drug in female urine, the relative proportion
of metabolites in total urinary radioactivity was lower in female rats.
For both genders, all metabolites accounting for >1% of total urinary
radioactivity are listed in table 4. The mean sum of all unidentified
metabolites was <1.8% for males and <0.5% for females.
A typical HPLC profile of urinary radioactivity is shown in fig. 4.
Fifteen urinary metabolites were tentatively identified, and MS data
were acquired for each metabolite. For the sake of brevity, we have
chosen to present the MS evidence only for the major urinary
metabolites. The data supporting the structure of each metabolite are
given below.
Metabolites 6 and 7. The protonated molecular ion for these metabolites
was m/z 557. Their respective HPLC retention times were 10.7 and 15.3 min. The structures were deduced from CID spectra, which were
consistent with cysteine conjugation plus hydroxylation. These cysteine
metabolites are proposed to be degradation products of GSH conjugates
of hydroxylated zopolrestat. A product-ion spectrum for the ion at
m/z 557 (fig. 5) provided
evidence for the proposed structure. The two metabolites showed similar
fragmentation spectra. The product-ion spectrum shows dehydration
(557 18 = 539) and neutral loss of 121 amu (cysteine
moiety; 557 121 = 436). In comparison with the product-ion
spectrum of zopolrestat (fig. 2), the metabolite product ions of
m/z 390 (374+O), 248 (216+S), and 232 (216+O) indicate that
the oxidation and conjugation of cysteine occur on the benzothiazole
moiety. Two chromatographic bands were present in the extracted ion
chromatogram and might be attributable to the existence of several
positional isomers for the proposed structure.
The in vivo formation of the GSH conjugates is thought to be
the result of cytochrome P450-mediated epoxidation, followed by
nucleophilic addition of GSH (Armstrong et al., 1981;
Baillie and Slatter, 1991; Buckpitt et al., 1987; Jones
et al., 1993; Nicoll-Griffith et al., 1993). The
sites of oxidation and conjugation on the benzothiazole ring may
change, resulting in positional isomers. Because the C6-C7 bond is the
least sterically hindered, it is suspected that epoxidation and GSH
addition would occur at C6 and C7 of the benzothiazole moiety.
Metabolites 8 and 9. The protonated molecular ion of these metabolites
was m/z 599. Their respective HPLC retention times were 16.5 and 16.7 min. The structures were deduced from CID spectra, which were
consistent with NAC (or mercapturic acid) conjugation plus
hydroxylation on the benzothiazole moiety. Evidence for this assignment
can be found in the product-ion spectrum for the ion at m/z
599 (fig. 6). The product ion at
m/z 232 suggests oxidation of the benzothiazole moiety, and
the product ion at m/z 162 represents the molecular ion of
NAC. Corroborating product ions are m/z 248 (216+S), 452 (420+S), and 581 (599H2O). Two chromatographic
bands were present in the extracted ion chromatogram and may be
attributable to the existence of several positional isomers for the
proposed structure.
These metabolites were most likely formed by the enzymatic activity of
N-acetyltransferase on metabolites 6 and 7. Isoforms of
N-acetyltransferase catalyze the transfer of an acetyl group from acetyl-CoA to the amino termini of most eukaryotic proteins (Kulkarni and Sherman, 1994). Acetylation of the
S-substituted cysteine conjugate is the final step in the
biosynthesis of mercapturic acid (Duffel and Jakoby, 1982; Tate, 1980).
Metabolite 10. The protonated molecular ion of this metabolite was
m/z 581. The HPLC retention time was 19.4 min. Its structure was deduced from the CID spectrum, which was consistent with NAC conjugation at the benzothiazole moiety. The product-ion spectrum of
the protonated molecular ion at m/z 581 is shown in fig.
7. Unlike metabolites 8 and 9, the mass
spectrum shows no product ion at m/z 162 representing the
NAC moiety. However, the immediate loss of an acetyl group (539 = 581 42) and the presence of an ion at m/z 452 (420+S) are indicative of the NAC conjugate. It is notable that the ion
at m/z 232 is absent from the spectrum but an ion at
m/z 248 (216+S) is present. These data indicate that the
sulfur atom is attached to the benzothiazole ring (216 + 32 = 248 and 374 + 32 = 406), but no oxidation occurs (216 + 16 = 232)
on that moiety. The chromatographic peak shape reveals a very narrow
bandwidth for this metabolite.
This metabolite may be a product of direct GSH conjugation without
epoxidation. It is known that hepatic GSH
S-alkenetransferase catalyzes an efficient reaction of the
xenobiotic material with GSH (Hutson, 1970). Substitution typically
occurs at an activated carbon atom, to the electronegative
functionality. It has been reported that benzothiazoles preferentially
undergo electrophilic substitution on the carbocyclic ring, principally
at C4 and C6 (Rance, 1989) (position 4 of this zopolrestat moiety is
expected to be the most electrophilic site). The location of the
CF3 group on the zopolrestat molecule fulfills
all of the aforementioned criteria to accommodate GSH conjugation to
the carbocyclic ring of the benzothiazole moiety. Another possible
origin for this metabolite could involve the in vivo
dehydration of metabolite 8 and/or metabolite 9. Dehydration may also
have occurred in vitro, after the addition of acid to the
urine during sample collection and extraction.
Metabolite 12. The protonated molecular ion of this metabolite was
m/z 248; its HPLC retention time was approximately 23 min. Weak responses were observed in the positive-ion mode, and the structure of this metabolite was deduced from a parent-ion scanning experiment for the ion at m/z 204. Additional evidence was
obtained from a Q1 experiment and product-ion scanning of the ion at
m/z 265 (the ammoniated molecular ion). Because the
positive-ion conditions yielded poor sensitivity, negative ion-spray
conditions were used. With negative ion-spray ionization, an intense
response was observed at the retention time of the radioactive
metabolite, and a molecular ion of m/z 306 was deduced to be
the acetate adduct molecular ion. Its product-ion spectrum is shown in
fig. 8 and supports the proposed
structure.
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Fig. 8.
CID product-ion spectrum for the acetate
adduct molecular ion at m/z 306 for urinary metabolite
12.
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Unchanged Drug. Nonmetabolized zopolrestat eluted at a retention time
(approximately 24.5 min) identical to that of an authentic standard,
showed the expected molecular ion of m/z 420 at the time of
radioactivity elution, and demonstrated a matching CID product-ion spectrum.
Identification of Biliary Metabolites.
Bile samples from eight rats (four of each gender) were analyzed
by simultaneous MS and -RAM detection. The amount of radioactivity associated with each metabolite or drug peak was quantified. A typical
profile of biliary radioactivity is shown in fig.
9, and table
5 provides a summary profile of
radioactivity percentages for each bile sample. In male rats, unchanged
zopolrestat was the major radioactive component (73.1%), and
metabolite 18 was the next most abundant component (12.2%). In female
rats, metabolite 18 was the major radioactive component (56.3%), and
unchanged zopolrestat was the next most abundant form of radioactivity
(34.0%). The contribution of all other metabolites was <15% of total
drug-related material in the analyzed samples.
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Fig. 9.
Representative HPLC radiochromatograms of
male (top) and female (bottom) rat
biliary metabolites.
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As with the urinary metabolites, we have chosen to present the MS
evidence only for the major biliary metabolites. The data supporting
the structure of each metabolite are given below.
Metabolite 2. The protonated molecular ion of this metabolite was
m/z 743, and its HPLC retention time was approximately 3 min. Its structure was deduced from the CID spectrum of the ion at
m/z 743, which was consistent with GSH conjugation plus
hydration of the benzothiazole moiety of zopolrestat. MS evidence
was found in neutral-loss scanning of 129 amu and in the product-ion
spectrum for the ion at m/z 743 (fig.
10). The product ion at m/z
306 represents the GSH adduct, and the product ions at m/z
130 and 177 represent the secondary fragmentation products of the GSH
adduct. The molecular ion of zopolrestat (m/z 420) is
present in conjunction with other commonly seen product ions that
indicate conjugation with the sulfur atom of GSH (406 = 374 + S;
for m/z 449, see proposed structure in fig. 10).
Metabolite 3. The protonated molecular ion of this metabolite was
m/z 686, and its HPLC retention time was approximately 4 min. Its structure was deduced from the CID spectrum of the ion at
m/z 686, which was consistent with formation of a
glutamyl-cysteine conjugate plus hydroxylation of the benzothiazole
moiety. The product-ion spectrum for the ion at m/z 686 is
shown in fig. 11. Supporting evidence
can be found in the product ion at m/z 249, representing the
glutamyl-cysteine moiety. The product ions at m/z 406/408,
449, 495, and 539 represent different stages of fragmentation. Because
of the similar elution volumes of metabolites 2 and 3, the
radioactivity associated with these metabolites was combined.
Metabolite 10. This metabolite had been previously identified in rat
urine. The molecular ion of this metabolite was m/z 581, and
its HPLC retention time was approximately 19.5 min. MS evidence was
gathered from neutral-loss scanning of 129 amu and parent-ion scanning
of m/z 452. The CID spectrum for the ion at m/z
581 for the biliary metabolite matches the CID spectrum for the ion at m/z 581 for the urinary metabolite (fig. 7).
Metabolite 18. The protonated molecular ion of this metabolite was
m/z 596. Several partially resolved chromatographic peaks exhibited HPLC retention times between 23 and 26 min (fig. 9). Its
structure was deduced from the CID spectrum for the ion at m/z 596, which was consistent with an acyl glucuronide of
zopolrestat. The product-ion spectra of the chromatographic peaks,
although not identical in ion ratios, were nearly identical in ion
masses. A common product-ion spectrum is shown in fig.
12. The initial loss of 176 amu
indicates the presence of a glucuronic acid conjugate. All product ions
of less than m/z 420 were identical to those in the
unchanged zopolrestat spectrum.
The presence of several chromatographic peaks for this mass may be
explained by the formation of different acyl glucuronide isomers
(i.e. 2'-, 3'-, or 4'-O-acyl). Rearrangement of
the acyl glucuronide conjugate by intramolecular transesterification at the hydroxyl groups of the glucuronic acid moiety has been reported (Akira et al., 1997; Benet and Spahn, 1988; Faed, 1984; Lenz
et al., 1996). The stabilities of these conjugates are
reportedly pH dependent, and acid stabilization has been recommended
(Benet and Spahn, 1988). It was shown in previous studies that the acyl glucuronides of zopolrestat could be hydrolyzed under alkaline conditions, so that concentrations of the aglycone could be determined (Inskeep et al., 1991). The product-ion spectra from these
biliary chromatographic peaks were similar, and the product ions of
less than m/z 420 were the same as those of unchanged
zopolrestat. These data suggest that the drug molecule is unchanged,
with the exception of the acyl glucuronidation.
Unchanged Drug. Nonmetabolized zopolrestat eluted at a retention time
identical to that of an authentic standard, exhibited the expected
molecular ion of m/z 420 at the time of radioactivity elution, and yielded a matching CID product-ion spectrum.
| |
Discussion |
Tissue Distribution Study.
The values for Cmax and the time of
Cmax reported for zopolrestat in plasma and
tissues in this study should be regarded as approximations, because of
the long intervals between samplings. However, in a previous
pharmacokinetic study of zopolrestat in rats dosed orally at 50 mg/kg
(Inskeep et al., 1991), plasma and tissue (nerve, kidney,
and lens) samples were collected at more frequent intervals in the
early phase after dosing (0.5, 1, 2, 4, 8, and 12 hr) than in the
current study, and these results were used to guide sampling time
selection in the current study. In general, the times of
Cmax were similar for the two studies, although Cmax values were slightly higher
in the previous study.
The half-life of radioactivity in most tissues (8-10 hr) was similar
to the half-life in blood. The longest half-life, 46 hr, was for
testes, whereas liver, skin, and sciatic nerve had intermediate
half-lives of approximately 20 hr. Although the half-life in testes was
relatively long, total exposure to radioactivity, as expressed by
AUC0-, was low, compared with exposure in
most of the other tissues (table 1).
Tissue levels of radioactivity were below the lower limit of
quantification (generally representing <2% of maximal tissue levels
of radioactivity) for all tissues except testes, skin, sciatic nerve,
liver, and intestines by 96 hr after dosing. Only testes (1.4% of the
maximal level) and liver (1.6% of the maximal level) had quantifiable
levels of radioactivity remaining at 168 hr after dosing. No
radioactivity could be quantified at 48 hr after dosing (<12% of
maximal levels) in eye, brain, and adrenal glands. By 168 hr after
dosing, radioactivity represented <2% of maximal tissue levels for
all tissues except eye, brain, sciatic nerve, adrenal glands, and fat
(for which the amount of tissue available for assay was limiting). For
these latter tissues, the limit of quantification at 96 hr after dosing
represented 4-20% of maximal tissue levels, and, assuming a half-life
of approximately 10 hr for these tissues, 
1% of maximal tissue
levels would be expected at 168 hr after dosing. Compared with the
maximal level of radioactivity, which was generally attained at 4 hr
after the dose, no tissue contained >2% of its maximal level by the
end of the study at 168 hr after the dose.
Bile Duct-Cannulated Rat Study.
The greater recovery of administered radioactivity in male rats than in
female rats was most likely the result of better bile duct cannula
patency over the 4-day period in males. Most of the radioactive dose
was recovered in the bile, indicating that biliary elimination is the
major route of elimination for zopolrestat and/or its metabolites. In
both genders, approximately 15% of the dose was recovered in feces,
indicating that 
85% of the dose of zopolrestat was absorbed. Urinary
elimination of radioactivity was slightly greater in females. The
greater urinary elimination of radioactivity in females represents a
significant gender effect on the disposition of zopolrestat in rats. In
multiple-oral dose studies in Sprague-Dawley, Fischer, and Long-Evans
rats, using doses of 50 or 100 mg/kg/day, systemic exposure (AUC and
approximate Cmax) was consistently 2-fold
higher in males than in females (data not shown).
The proposed metabolic scheme for zopolrestat in rat urine and bile is
depicted in fig. 3. A major portion of orally administered zopolrestat
was excreted as unchanged drug in urine (females) and bile (males and females).
Fifteen metabolites of zopolrestat were identified in a male rat urine
sample. For male rats, most of the radioactivity in urine was
attributable to the degradation products of GSH conjugates of
hydroxylated zopolrestat (metabolites 6-9). Parent drug was the second
most abundant form of radioactivity in urine. Benzothiazole carboxylic
acid (metabolite 12) accounted for approximately 10% of total urinary
radioactivity. In addition, approximately 10% of urinary radioactivity
was present as the mercapturic acid and/or ether glucuronide conjugates
(metabolites 10 and 11, respectively), which were not
chromatographically resolved. In female rat urine, the same metabolites
were observed in approximately the same amounts, but the relative
proportions in urine were lower because of the excess of unchanged drug
in females. For both genders, only metabolites representing >1% of
total urinary radioactivity are shown in table 4. All GSH-related
metabolites accounted for only 7.8 and 2.1% of the administered dose
in male and female rats, respectively. Female rats excreted more
radioactivity in urine than did male rats, and most of the increased
material was attributed to unchanged drug. The amounts and proportions
of the urinary metabolites were similar for males and females. It is
noteworthy that, among the minor metabolites whose radioactive
contributions were not quantifiable, five single-oxidation products
were identified. The metabolic characteristics of all of the minor
metabolites have been summarized in table 3.
Eight metabolites of zopolrestat were identified in rat bile samples,
of which four were also identified in rat urine. Parent drug and acyl
glucuronidation products accounted for >85% of the identified
radioactivity in male and female bile samples. Other minor metabolites
included products of GSH conjugation and subsequent degradation of the
GSH moiety (<9.5% of the administered dose).
In conclusion, the pharmacokinetics of zopolrestat in rats primarily
involve biliary and urinary excretion of parent drug and/or its acyl
glucuronide. Several oxidative pathways exist but are quantitatively
much less important in the overall disposition of zopolrestat.
Gender-related differences in renal elimination have been associated
with higher systemic exposure in male rats than in female rats.
We thank Kim M. Davis and David N. Guzzi for expert technical
assistance in these studies and B. Mylari and W. Zembrowski for
synthesis of the putative metabolites.
Abbreviations used are:
LSA, liquid
scintillation analysis;
CID, collisioninduced dissociation;
ACN, acetonitrile;
NH4OAc, ammonium acetate;
-RAM, -radioactivity monitor;
NAC, N-acetylcysteine;
Cmax, maximal plasma concentration;
GSH, glutathione.
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Abstract
Introduction
-acetyltransferase in the yeast Saccharomyces cerevisiae.
J Biol Chem
269:
13141-13147
