sábado, 20 de marzo de 2010


SB-209247 [(E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic
acid], an anti-inflammatory leukotriene B4 receptor antagonist, was
associated in beagle dogs but not male rats with an inflammatory
hepatopathy. It also produced a concentration-dependent (10-1000 μM)
but equal leakage of enzymes from dog and rat precision-cut liver
slices. The hepatic metabolism of SB-209247 was investigated with
reference to the formation of reactive acyl glucuronides.
[14C]SB-209247 (100 μmol/kg) administered i.v. to anesthetized male
rats was eliminated by biliary excretion of the acyl glucuronides of
the drug and its sulfoxide. After 5 h, 1.03 ± 0.14% (mean ± S.E.M., n
= 4) of the dose was bound irreversibly to liver tissue. The sulfoxide
glucuronide underwent pH-dependent rearrangement in bile more rapidly
than did the SB-209247 conjugate. [14C]SB-209247 was metabolized by
sulfoxidation and glucuronidation in rat and dog hepatocytes, and
approximately 1 to 2% of [14C]SB-209247 (100 μM) became irreversibly
bound to cellular material. [14C]SB-209247 sulfoxide and glucuronide
were the only metabolites produced by dog, rat, and human liver
microsomes in the presence of NADPH and UDP-glucuronic acid (UDPGA),
respectively. Vmax values for [14C]SB-209247 glucuronidation by dog,
rat, and human microsomes were 2.6 ± 0.1, 1.2 ± 0.1, and 0.4 ± 0.0
nmol/min/mg protein, respectively. Hepatic microsomes from all three
species catalyzed UDPGA-dependent but not NADPH-dependent irreversible
binding of [14C]SB-209247 (100-250 μM) to microsomal protein. Although
a reactive acyl glucuronide was formed by microsomes from every
species, the binding did not differ between species. Therefore,
neither the acute cellular injury nor glucuronidation-driven
irreversible protein binding in vitro is predictive of the
drug-induced hepatopathy.
Previous SectionNext SectionNumerous drugs containing a carboxylic
acid group, and especially certain nonsteroidal anti-inflammatory
drugs (NSAIDs), have been associated with diverse clinical toxicities
(Griffin and Scheiman, 2001). Thus hepatotoxicity, to divergent
extents, is known for nearly all NSAIDs, although the proportion of
hepatic versus nonhepatic side effects varies considerably between the
drugs and hepatotoxicity is not exclusive to the carboxylate compounds
(Merlani et al., 2001). The liver damage is in most cases considered
to derive from an idiosyncratic metabolic and/or immune reaction that
is essentially independent of dose (Boelsterli, 2002; Bailey and
Dickinson, 2003). Some carboxylate drugs are also linked with rarer
hypersensitivity reactions. In general, the symptomatic hepatotoxicity
is uncommon, mild, and reversible; only in exceptional instances does
it result in fulminant liver failure.
SB-209247 [(E)-3-[6-[[(2,6-dichlorophenyl)-thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic
acid; Fig. 1], an LTB4 receptor antagonist with oral anti-inflammatory
activity in mice (Daines et al., 1996; Davis et al., 2000), was
developed for the treatment of dermal inflammatory diseases. LTB4 is a
potent, proinflammatory neutrophil activator, chemotactic agent, and
apoptosis regulator that is released from neutrophils and other
leukocytes during physiological and pathophysiological inflammatory
reactions (Crooks and Stockley, 1998). Development of SB-209247 was
discontinued when administration to beagle dogs at 60 to 1000
mg/kg/day (0.13-2.17 mmol/kg/day) for 28 days was associated with
non-dose-dependant inflammatory hepatopathy, with minimal
hepatocellular necrosis being observed in the more severe cases
(GlaxoSmithKline, Uxbridge, Middlesex, UK, unpublished data). Male
rats were given doses of SB-209247 that produced maximum plasma
concentrations similar to those attained in the dogs, but no
pathological changes were found in their livers. The etiology of the
hepatopathy in dogs is unknown: an influx of inflammatory cells is a
common response to hepatocellular injury, although it is superficially
paradoxical in this case because LTB4 receptor antagonists inhibit,
particularly, the chemotaxis of neutrophils and, inter alia, their
accumulation in skin (Brooks and Summers, 1996). Hepatic adverse
reactions attributable to the various types of leukotriene antagonist
are not unknown in experimental animals and humans, but there is
presently no clear evidence for a generic association related to
either pharmacological activity or the presence of a carboxylic acid
function (Fretland et al., 1995; Chambers et al., 1999; Reinus et al.,
Several mechanisms have been proposed by which a carboxylic acid might
cause hepatotoxicity (Boelsterli, 2002). Some carboxylic acid drugs
have the potential to initiate cell injury by acting directly on
mitochondria (Boelsterli, 2003). Additional cytotoxic actions might
require bioactivation, mediated by either oxidative metabolism (Bort
et al., 1999) or the conjugation reactions forming acyl-CoA thioesters
and acyl glucuronides (Boelsterli, 2002; Bailey and Dickinson, 2003).
Ester-linked glucuronides have received particular attention because
of their well documented ability to react with cellular proteins and
DNA through nucleophilic addition or displacement. This adduction can
result in perturbation of molecular and organellar activity (Sallustio
and Holbrook, 2001) or the generation of neoantigens, which instigate
immune-mediated cytotoxicity (Bailey and Dickinson, 2003). The
cytotoxic or cytostatic actions of acyl glucuronides have in certain
cases suggested direct effects of the conjugate (Sallustio et al.,
1997; Seitz and Boelsterli, 1998; Bailey and Dickinson, 2003).
Conjugation with d-glucuronic acid is a common route of elimination
for carboxylic acids and is catalyzed by a number of hepatic UGTs
(Soars et al., 2001). Biosynthetic (β-1-O-) acyl glucuronides (Bailey
and Dickinson, 2003) are generically reactive, electrophilic compounds
capable of undergoing facile hydrolysis to the agylcone, rearrangement
via reversible acyl migration and subsequent mutarotation, and
covalent binding to proteins (King and Dickinson, 1991; Corcoran et
al., 2001; Kenny et al., 2004). However, there is considerable
variation in the intrinsic stability and reactivity of such conjugates
(Boelsterli, 2002). Protein adducts are formed either by nucleophilic
displacement of the glucuronic acid moiety, which produces
aglycone-protein combinations, or by a glycation pathway, in which
intramolecular migration of the acyl residue from C-1 allows opening
of the glucuronic acid ring to create an aldehyde intermediate and
subsequent formation of glucuronide-protein adducts (Bailey and
Dickinson, 2003).
The propenoic acid moiety of SB-209247 and the absence of conspicuous,
alternative conjugable groups make this drug a potential substrate for
extensive acyl glucuronidation in vivo. We have considered the
possibility that it might be bioactivated in dogs via this pathway:
there are instances of carboxylic acid drugs undergoing
species-selective acyl glucuronidation by hepatic microsomes (Soars et
al., 2001; Prueksaritanont et al., 2002). The metabolism of SB-209247
and the compound's hepatocytotoxicity in vitro have been investigated
with particular reference to comparative rates of glucuronidation and
covalent binding to hepatocellular proteins.
Previous SectionNext SectionMaterials and Methods
Chemicals. NADPH, sodium estrone β-d-glucuronide, UDPGA, and H-2
β-glucuronidase-arylsulfohydrolase (Helix pomatia) were obtained from
Sigma-Aldrich (Poole, Dorset, UK). Williams' medium E, Waymouth's MB
752/1 medium, hepatocyte-grade (type IV) collagenase, and other cell
culture reagents were obtained from Invitrogen (Paisley, Scotland,
UK). SB-209247 (Daines et al., 1996), SB-209247 sulfoxide (SB-215244),
and [14C]SB-209247 (41.5 mCi/mmol; radiochemical purity by HPLC,
99.5%) were synthesized at GlaxoSmithKline (King of Prussia, PA).
HPLC-grade solvents were products of Fisher Scientific (Loughborough,
Leicestershire, UK). All other chemicals were purchased from BDH
(Poole, Dorset, UK).
Animals. Adult male Wistar rats were obtained from a breeding colony
in the Biomedical Services Unit, The University of Liverpool. The
adult male Sprague-Dawley rats and beagle dogs used for the in vitro
toxicity (liver slices) study were bought from Charles River
Laboratories, Inc. (Wilmington, MA) and Marshall Farms (North Rose,
NY), respectively; they were maintained at GlaxoSmithKline (King of
Prussia, PA). The beagle dogs used for hepatocyte preparation were
obtained from Huntingdon Life Sciences (Cambridgeshire, UK) and were
maintained at GlaxoSmithKline (The Frythe, Hertfordshire, UK).
Human Livers. Histologically normal livers were obtained from four
white male transplant donors (aged 19-41 years). Approval for the
study was granted by the relevant ethical committees, and consent was
obtained from the donors' relatives. The certified cause of death was
either cerebrovascular injury or traumatic injury consequent to a road
traffic accident. The livers were removed and transported from the
hospital to the laboratory within 30 min of death. They were then
portioned, frozen in liquid nitrogen, and stored at -80°C.
Metabolism of [14C]SB-209247 in the Rat. Adult male Wistar rats
(200-250 g) were anesthetized with urethane (1.4 g/ml in isotonic
saline; 1.0 ml/kg i.p.) and cannulated via the trachea, jugular vein,
and common bile duct. [14C]SB-209247 (100 μmol/kg; specific activity
0.069 μCi/μmol) freshly dissolved in dimethyl sulfoxide (200 μl) was
injected intravenously over 10 min. Bile was collected as hourly
fractions for 5 h into preweighed Eppendorf tubes cooled on ice,
acidified with 4% (v/v; pH 1.8) orthophosphoric acid (10:1 bile/acid,
v/v), and protected from light in amber-glass vials (SB-209247 is
photolabile in daylight). Aliquots were either assayed for
radioactivity by liquid scintillation counting (10 μl, two times) or
analyzed by parallel LC-MS and radiochromatography (50 μl). The bile
was stored at -80°C. Enzymatic hydrolysis of glucuronides in bile
samples (200 μl) was achieved by incubation with H-2 preparation (20
μl; β-glucuronidase, 131 units/μl) in 0.1 M sodium acetate buffer (pH
5) at 37°C for 4 h. The hydrolysate was analyzed by radiometric HPLC.
Residues of radiolabeled material in tissues (brain, heart, kidney,
liver, lung, and spleen) were measured as described previously (Maggs
et al., 2000). Portions of liver (approximately 100 mg) were
homogenized in Hanks' balanced salt solution, protein was precipitated
from the homogenates with 3 volumes of acetonitrile (3 ml), and
radiolabeled material bound irreversibly to the pelleted protein (750g
for 15 min) was estimated by exhaustive solvent extraction as
described below.
Stability of Acyl Glucuronide Metabolites. Bile (0- to 1-h collection)
from a rat given [14C]SB-209247 (100 μmol/kg; specific activity 0.069
μCi/μmol) was adjusted to pH 5.0 or 7.4 with 0.1 M sodium phosphate
buffer and mixed with a solution of estrone glucuronide in water (200
μg/ml; 3:2 v/v). An aliquot of the resulting mixture was analyzed
immediately (time = 0) by LC-MS with selected ion monitoring of the
glucuronides of estrone (16.5 min), SB-209247 (32 min), and SB-209247
sulfoxide (32.5 min) at m/z 445, 634, and 650 ([M - 1]-),
respectively. The remainder was kept at 37°C. Samples were removed and
analyzed at intervals for 24 h. The mass chromatogram peak area for
each acyl glucuronide was expressed as the ratio metabolite/estrone
Metabolism of [14C]SB-209247 by Hepatocytes. Hepatocytes were isolated
from whole livers of adult male Wistar rats (180-200 g) by two-step
collagenase perfusion (Tettey et al., 1999) and from the cordate lobe
of adult male beagle dogs by a three-step perfusion technique derived
from the method of Strom et al. (1982). The viability of the cell
suspensions, typically ≥88% and ≥98%, respectively, was determined by
trypan blue exclusion.
Freshly isolated rat hepatocytes (2.4 × 106 viable cells/ml) suspended
in Krebs-Henseleit buffer (pH 7.4) were incubated with [14C]SB-209247
(final concentration, 100 μM; 1 μCi) dissolved in dimethyl sulfoxide
(final concentration, 0.1% v/v). The total volume was 5 ml.
Incubations were carried out on four separate occasions in rotating
50-ml round-bottom flasks at 37°C under an atmosphere of O2 and CO2
(95:5 v/v). [14C]SB-209247 was also incubated in the absence of
hepatocytes. After 1 or 3 h, ice-cold acetonitrile (15 ml) was added
to the incubations and they were placed on ice for 15 min. The
resulting precipitate was sedimented at 750g, and the unbound
radiolabeled material associated with it was extracted with
acetonitrile (3 ml, two times). The supernatant and extracts were
combined, evaporated to dryness under N2 at 40°C, and reconstituted in
acetonitrile (200 μl) for analysis by LC-MS and radiochromatography.
Freshly isolated dog hepatocytes in Williams' medium supplemented with
newborn calf serum (10% v/v), insulin (0.1 μg/ml), and antibiotics
(penicillin, streptomycin, and neomycin sulfate; each 100 μg/ml) were
seeded in six-well collagen-coated plates (5 × 105 cells/ml, 3 ml).
They were allowed to adhere under an atmosphere of O2 and CO2 (95:5
v/v) at 37°C. The medium was changed to serum- and insulin-free
Williams' medium, and the cells were incubated with [14C]SB-209247
(final concentration, 100 μM; 0.6 μCi) dissolved in dimethyl sulfoxide
(final concentration, 0.1% v/v) for 4 h. Incubations were terminated
with ice-cold acetonitrile (12 ml) and acidified with 17% (v/v)
orthophosphoric acid (1:10 v/v). The culture medium was removed and
discarded. The hepatocytes were recovered from the well plate with a
cell scraper, left on ice for 15 min, and centrifuged. The supernatant
and two acetonitrile extracts (3 ml) of the cell pellet were combined,
evaporated to dryness under N2 at 40°C, and reconstituted in
acetonitrile (200 μl) for analysis. The cell pellets from both sets of
incubations were retained for determination of irreversibly bound
radiolabeled material.
Toxicity of SB-209247 to Rat and Dog Liver Slices. Precision-cut liver
slices were prepared from one male beagle dog (10.5 kg) and two male
Sprague-Dawley rats. The dog was anesthetized with acepromazine and
pentobarbital and then exsanguinated. The rats were euthanized with
carbon dioxide and exsanguinated. Livers were kept in ice-cold
Krebs-Henseleit buffer (pH 7.4) until they were processed. Briefly,
8-mm cores produced with a stainless steel coring tool were sliced
with a Krumdieck precision slicer (Krumdieck et al., 1980). Slices
(approximately 250 μm thick, 11-15 mg wet weight) were collected,
placed individually upon stainless steel mesh screens, and incubated
in Waymouth's medium containing 25 mM HEPES and 10 mM glucose in a
rolling incubator under an atmosphere of O2 and CO2 (95:5 v/v) at
37°C. Slices were incubated in prewarmed medium (1.7 ml) for 1 h to
equilibrate. This medium was replaced with prewarmed medium (1.7 ml)
containing either dimethyl sulfoxide (1% v/v), SB-209247 in dimethyl
sulfoxide (final drug concentration, 10 μM, 100 μM, or 1 mM), or
acetaminophen dissolved directly in medium (final concentration, 27 or
90 mM). Slices were incubated in duplicate for 2, 4, or 24 h, after
which the medium was removed and assayed for LDH leakage
spectrophotometrically (Bergmeyer and Bernt, 1974). Each slice was
sonicated in phosphate-buffered saline (pH 7.4; 1 ml) on ice for 30 s
to liberate the remaining LDH.
Preparation of Microsomes. Livers were removed from adult male Wistar
rats immediately after they were killed by cervical dislocation and
homogenized individually in 2 volumes of ice-cold 67 mM potassium
phosphate buffer (pH 7.5) containing 0.15 M potassium chloride.
Samples (10-20 g) of frozen human and dog liver stored at -80°C were
also homogenized individually. Microsomal fractions were prepared
according to the method of Gill et al. (1995). Protein concentrations
were determined by the method of Lowry et al. (1951). Equal amounts of
protein from four rat, dog, or human livers were pooled.
Microsomal Oxidation of [14C]SB-209247. Incubations were carried out
in 4-ml amber-glass vials. [14C]SB-209247 (final concentration, 100 μM
or 250 μM; 0.2 μCi) dissolved in dimethyl sulfoxide (1 μl) was added
to microsomes (1 mg of protein) in 67 mM phosphate buffer (pH 7.5)
containing 10 mM MgCl2 to give a final volume of 1 ml. Following
preincubation at 37°C for 2 min in a shaking water bath, the reaction
was initiated by addition of NADPH (final concentration, 1 mM). NADPH
was omitted from control incubations. After 30 min, the reaction was
terminated with ethyl acetate (3 ml). The supernatants of two 15-min
extractions of the protein pellet (750g for 15 min) were pooled and
evaporated to dryness under a stream of N2 at 40°C. The residue was
reconstituted in acetonitrile (300 μl) and stored at -80°C until
analyzed by radiometric HPLC and/or LC-MS. The protein pellets were
retained for determination of irreversibly bound radiolabeled
Microsomal Glucuronidation of [14C]SB-209247. Incubations were carried
out in 4-ml amber-glass vials. [14C]SB-209247 (final concentration,
100 μM or 250 μM; 0.2 μCi) dissolved in dimethyl sulfoxide (1 μl) was
added to microsomes (1 mg of protein) in 50 mM Tris-HCl buffer (pH
7.5) containing 10 mM MgCl2 and Brij 58 surfactant (0.1 mg/ml).
Following preincubation at 37°C for 2 min in a shaking water bath, the
reaction was initiated by addition of UDPGA (final concentration, 3
mM). The final volume was 1 ml. UDPGA was omitted from control


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