In Vitro Metabolic Characterization, Phenotyping, and Kinetic
Studies of 9cUAB30, a Retinoid X Receptor-Specific Retinoid
Gregory S. Gorman, Lori Coward, Corenna Kerstner-Wood, Lea Cork, Izet M. Kapetanovic,
Wayne J. Brouillette, and Donald D. Muccio
Southern Research Institute, Birmingham, Alabama (G.S.G., L.C., C.K.-W., L.C.); Chemoprevention Agent Development
Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, Maryland (I.M.K.); and Department of
Chemistry, the University of Alabama at Birmingham, Birmingham, Alabama (W.J.B., D.D.M.)
Received November 21, 2006; accepted April 16, 2007
The present study was conducted to compare the in vitro phase I
and phase II metabolic profiles of (2E,4E,6Z,8E)-8-(3?,4?-dihydro-
(9cUAB30) in human, rat, and dog microsomes and to characterize
and identify the associated metabolic kinetics and specific
isozymes from human liver microsomes (HLM) responsible for
metabolism, respectively. Data from these experiments revealed
that nine (M1–M9) phase I metabolites along with a single glucuro-
nide conjugate were observed across the species investigated.
With the exception of glucuronidation, no evidence of metabolism
was detected for phase II enzymes (data not shown). Significant
differences between species with regard to metabolic profile, sta-
bility, and gender were noted. For the eight phase I metabolites
detected in HLM, the specific isozymes responsible for the bio-
transformations were CYP2C8, CYP2C9, and CYP2C19, with minor
contributions from CYP1A2 and CYP2B6. For the glucuronide con-
jugate, UGT1A9 was the major catalyzing enzyme, with a minor
contribution from UGT1A3. Kinetic analysis of eight of the detected
metabolites indicated that four seemed to follow classical hyper-
bolic kinetics, whereas the remaining four showed evidence of
either autoactivation or substrate inhibition.
Retinoids belong to a class of compounds chemically related to
vitamin A and have been shown to be effective in vitro against many
types of cancer, including breast cancer (Crouch and Helman, 1991;
Delia et al., 1993; Rubin et al., 1994; Sun et al., 1997; Wu et al.,
1997). Biological activities of retinoids are mediated through retinoid
X receptors (RXR), which belong to a family of transcription factors
known as hormone nuclear receptors. These receptors mediate the
effects of hydrophobic hormones on gene transcription. When acti-
vated by retinoids, RXR can modulate several signaling pathways
through its ability to form heterodimers with other members of the
receptor family. 9cUAB30 (Fig. 1) has been shown to be efficacious
alone or in combination therapy with tamoxifen in the prevention of
mammary cancers (Grubbs et al., 2003).
Integral with the drug development process is the importance to
characterize the metabolic profile of xenobiotics (Lin and Lu, 1997;
Pelkonen et al., 2005). For numerous drugs, the duration and intensity
of action are determined by their rate of metabolism from hepatic or
intestinal phase I [mainly cytochrome P450 (P450)] and phase II
[mainly UDP glucuronosyltransferases (UGT)] enzymes. Character-
izing these properties is critical to assessing viability and predicted
success of new drugs.
In this study, which represents the first in vitro metabolic investi-
gation of this compound in preclinical development, we investigate
the phase I and phase II metabolic properties of 9cUAB30 by hepatic
(human, rat, and dog) microsomes. Enzyme mapping studies using
recombinant P450 enzymes along with human liver microsomes
(HLM) were used to determine individual isozymes responsible for
metabolism along with their associated kinetics.
Materials and Methods
Chemical and Biological Reagents. 9cUAB30 was supplied by the Na-
tional Cancer Institute (Bethesda, MD), and ?-phenylcinnamic acid (?97%) as
the internal standard, 3?-p-hydroxypaclitaxel, paclitaxel, acetaminophen, and
acetaminophen-glucuronide were purchased from Sigma Chemical Co. (St.
Louis, MO). Ammonium acetate, acetonitrile, and methanol (high-perfor-
mance liquid chromatography grade) were obtained from Fisher Scientific
(Atlanta, GA). The NADPH regenerating system (solution A and B), UGT
assay mix (solution A and B), and 6?-hydroxy-paclitaxel were obtained from
BD Biosciences (Bedford, MA).
Microsomes and P450 Enzymes. All the species of hepatic microsomes
were obtained from XenoTech, LLC (Lenexa, KS). Isozymes CYP1A1,
CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1,
CYP3A4, and CYP4A11 (Bactosomes) obtained from Escherichia coli-ex-
pressed recombinant enzymes were purchased from Xenotech, LLC. UGT1A1,
UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10,
UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were purchased from BD
Metabolic Incubations. The incubations for metabolite identifications were
conducted with a 10 ?M concentration of the substrate (9cUAB30) in a total
publicationdate,andcitation information can befound at
ABBREVIATIONS: RXR, retinoid X receptor(s); 9cUAB30, (2E,4E,6Z,8E)-8-(3?,4?-dihydro-1?(2?H)-naphthalen-1?-ylidene)-3,7-dimethyl-2,4,6-oc-
tatrienoic acid; P450, cytochrome P450; UGT, UDP glucuronosyltransferase(s); HLM, human liver microsome(s); LC/MS/MS, liquid chromatog-
raphy/tandem mass spectrometry; RLM, rat liver microsome(s); DLM, dog liver microsome(s).
DRUG METABOLISM AND DISPOSITION
U.S. Government work not protected by U.S. copyright
DMD 35:1157–1164, 2007
Vol. 35, No. 7
Printed in U.S.A.
reaction volume of 0.5 ml at 37°C in a shaking water bath. A 20 ?M
concentration was used for phenotyping studies, whereas 0.2, 0.5, 1, 5, 10, 50,
and 100 ?M concentrations were used in the kinetic studies. The composition
of the reaction mixtures consisted of a pH 7.4 phosphate buffer solution
containing an NADPH regenerating solution consisting of 1.3 mM NADP?,
3.3 mM glucose 6-phosphate, 0.4 U/ml glucose-6-phosphate dehydrogenase,
120 ?M sodium citrate buffer, 3.3 mM magnesium chloride, and liver micro-
somes (1 mg/ml total protein) or isozymes (2.2–13.3 nmol/ml P450). The total
organic solvent concentration in the reaction mixture was approximately 0.6%
(v/v) and determined not to interfere with metabolic activity. For the glucu-
ronidation reactions, 9cUAB30 (10 ?M) was incubated with liver microsomes,
UDP-glucuronic acid cofactor at 2 mM in deionized water, along with 50 mM
Tris-HCl, 8 mM MgCl2, and 25 ?g of alamethicin in deionized water. The
reaction mixture also contained the NADPH regenerating solution as described
previously. Aliquots (50 ?l) from the reaction mixtures used to determine
metabolic stability were sampled at 5, 15, 30, 60, and 120 min, and the reaction
was quenched by the addition of an equal volume of acetonitrile. Reactions for
the kinetic and isozyme phenotyping studies were quenched after 15 min and
1 h of incubation time, respectively, with a volume of acetonitrile equal to that
of the reaction mixture. All the samples from the various reaction mixtures
were centrifuged at 11,000 rpm for 5 min, and the supernatant was analyzed
directly by liquid chromatography/tandem mass spectrometry (LC/MS/MS). A
series of positive and negative controls were simultaneously incubated under
identical conditions. The positive control substrate/metabolite pairs consisted
of paclitaxel/6?-hydroxypaclitaxel and 3?-p-hydroxypaclitaxel for oxidation
reactions along with acetaminophen/acetaminophen-glucuronide for glucu-
ronidation. Negative control reactions consisted of a reaction mixture with no
substrate and a separate reaction with no microsomes where quenched micro-
somes were back-added to the reaction mixture after incubation.
Analytical Method. Chromatographic separation of the metabolites was
achieved using a PerkinElmer Series 200 high-performance liquid chromatog-
raphy system (Norwalk, CT) with a 100 ? 2 mm, Aquasil C18reverse-phase
column (Thermo Electron, Bellefonte, PA). A mobile phase consisting of 5
mM ammonium acetate and methanol was optimized for the separation of the
metabolites. A gradient profile was used where the methanol concentration was
increased from 40% to 90% at a linear rate over 11.5 min and then back to 40%
in 2.5 min at a constant flow rate of 400 ?l/min. Mass detection was accom-
plished from 10-?l injections with an Applied Biosystems (Foster City, CA)
4000 QTRAP triple quadrupole ion trap mass spectrometer equipped with an
electrospray ionization source operated at a potential of 5 kV at 450°C. Data
were collected from the instrument using Analyst 1.4.1 (Applied Biosystems)
in both Q1 mass scan mode and independent data acquisition mode using
enhanced product ion scans in the ion trap mode for both polarities.
Metabolism by HLM. Five metabolites (M1–M5) in the positive
ion mode, three metabolites (M6–M8) in the negative ion mode, and
one glucuronide conjugate were detected by LC/MS/MS analysis
from the in vitro reaction medium after incubation of 9cUAB30 with
gender-specific HLM for 2 h at 37°C. Peaks corresponding to these
metabolites (Figs. 2–4) were absent in the negative control reactions
that were generated by adding solvent inactivated microsomes to the
reaction mixture after incubation. Metabolites M1 through M4 are
consistent with oxidized metabolites (M ? 16), whereas M5 was
found to be consistent with dehydrogenation/oxidation of the substrate
(M ? 14). Metabolites M6 through M8 were found to have masses
consistent with dehydrogenation (M ? 2). There was no evidence of
any conjugative phase II metabolism observed (data not shown).
Metabolism by Rat Liver Microsomes. In vitro reactions con-
ducted in rat liver microsomes (RLM) produced metabolites M1
through M8 and a single glucuronide conjugate as previously ob-
served in the HLM reactions. In addition to these, a new metabolite,
M9, corresponding to doubly oxidized species (M ? 32) was also
detected. There was no evidence of any conjugative phase II metab-
FIG. 1. Molecular structure of 9cUAB30.
FIG. 2. A–D, negative ion mode targeted
MRM transitions of phase I metabolites M1
through M5 of 9cUAB30 in male liver mi-
crosomes: human (A), dog (B), rat (C), and
negative control (D). IS, internal standard.
GORMAN ET AL.
olism observed (data not shown). Peaks corresponding to these me-
tabolites (Figs. 2–4) were absent in the negative control reactions.
Metabolism by Dog Liver Microsomes. In vitro reactions in dog
liver microsomes (DLM) resulted in detection of six previously ob-
served metabolites, M2 through M4 and M6 through M8, and a single
glucuronide conjugate as observed for both HLM and RLM. No
evidence for metabolites M1, M5, or M9 was found in reactions with
DLM. There was no evidence of any conjugative phase II metabolism
observed (data not shown). All the metabolites detected in these
reactions (Fig. 2–4) were absent in the negative control reactions.
Metabolic Stability. Metabolic stability of 9cUAB30 was deter-
mined in duplicate at five time points between 5 and 120 min in
gender-specific HLM, RLM, and DLM for both oxidative and glucu-
ronide-conjugate reactions. Figure 5, A through C, shows the phase I
metabolic stability for 9cUAB30 in both genders for rat, dog, and
human, respectively. Figure 6, A through C, shows the corresponding
stability of 9cUAB30 for the glucuronidation reactions. The error bars
reflect the difference of the individual values from the mean. These
data suggest that 9cUAB30 undergoes a significant amount of oxida-
tive metabolism in both genders of RLM with less than 15% of the
starting concentration (10 ?M) remaining after 120 min. Less signif-
icant losses of 9cUAB30 were observed in HLM and DLM. Under the
same incubation conditions, about 50% of the starting concentration
of 9cUAB30 remained in each gender of HLM, whereas 50 and 71%
remained in male and female DLM, respectively. For the glucuronida-
tion reactions, loss of 9cUAB30 was found to be rapid and extensive
in both genders of all three species such that 10% or less was found
to be remaining after 120 min of incubation.
FIG. 3. A–D, positive ion mode targeted
MRM transitions of phase I metabolites M6
through M8 of 9cUAB30 in male liver mi-
crosomes: human (A), dog (B), rat (C), and
negative control (D). IS, internal standard.
FIG. 4. A–D, negative ion mode targeted
MRM transitions of glucuronide metabo-
lites of 9cUAB30 in male liver micro-
somes: human (A), dog (B), rat (C), and
negative control (D).
IN VITRO METABOLIC CHARACTERIZATION OF 9cUAB30 IN MICROSOMES
Identification of Metabolites. Metabolite identification was based
on characteristic mass shifts of the molecular ions [M?H]?, [M-H]?
in combination with the product ions obtained from the enhanced
product ion mass spectrum of each peak in both polarity modes as
compared with an authentic 9cUAB30 standard. Characteristic ions
observed in the positive ion spectrum (Fig. 7A) for 9cUAB30 include
m/z 277 (loss of H2O), m/z 249 (loss of HCOOH), and m/z 235 (loss
of CH3COOH). Other abundant fragment ions include m/z 209
[M-86]?, m/z 195 [M-100]?, and m/z 169 [M-126]?, which likely
result from fragmentation of the alkene chain and contain the dihy-
dronaphthalen-1?-ylidene functionality. Fragment ions observed in the
negative ion spectrum (Fig. 7B) include m/z 249 (loss of CO2), m/z
181 [M-112]?, m/z 141 [M-152]?, and m/z 119 [M-174]?. In the
negative ion mode, M1 through M4 (m/z 309), M5 (m/z 307), and M9
(m/z 325) all had characteristic mass shifts of 16 Da (oxidation), 14
Da (oxidation/dehydrogenation), and 32 Da two degrees of oxida-
tion, respectively, for the parent and characteristic fragment ions.
Because of the limited amount of fragment ions detected for
metabolites M1 through M5, the precise location of each biotrans-
formation on the parent could not be made. In the positive ion
mode, metabolites M6 through M8 displayed mass shifts of ?2 Da
(dehydrogenation) for the parent and characteristic fragment ions.
Figure 8 shows the representative MS/MS spectra for M4, M5, and
M8. The single glucuronide metabolite was characterized by the M
? 176 ion and subsequent loss of 176 Da as detected in a neutral
loss scan (Fig. 9).
Isozyme Mapping. Commercially available human P450 and UGT
isoforms were evaluated for their ability to metabolize 9cUAB30. The
experiments were conducted by incubating the substrate with individ-
ual isozymes or UGT and monitoring the reaction mixture for the
previously described metabolites. The results for metabolites M1
through M5, M6 through M8, and the UGT are shown in Fig. 10. In
general, CYP2C8, CYP2C9, and CYP2C19 were determined to be
responsible for the majority of the observed biotransformations for the
oxidative metabolites M1 through M5, along with minor contributions
from CYP1A2. In particular, CYP2C9 was found to be the largest
FIG. 5. A–C, metabolic stability of 9cUAB30 in male (?) or female (f) liver
microsomes. Error bars on the graphs show the difference of the individual values
from the mean; rat (A), dog (B), and human (C).
FIG. 6. A–C, glucuronidation reactions in male (?) or female (f) liver micro-
somes. Error bars on the graphs show the difference of the individual values from
the mean; RLM (A), DLM (B), and HLM (C).
GORMAN ET AL.
single contributor to M1, whereas CYP2C8’s largest contribution was
to M3. CY2C19 contributed most significantly to M3 and M4,
whereas CYP1A2’s largest contribution was to M4. The only signif-
icant contribution to M5 appeared to come from CYP2C8. The group
of isozymes as found with M1 through M5 metabolites was also
determined to be the most active in the formation of the dehydroge-
nated metabolites M6 through M8. The formation of M6 was found to
be primarily from CYP2C9 with small contributions from CYP2C8,
CYP2C19, and CYP2B6, whereas M7 was produced almost equally
by CYP2C8 and CYP2C19. In the formation of M8, CYP2C19 was
observed as the single largest contributor; however, other contribu-
tions were also made by CYP2C8 and CYP2C9, along with minor
contributions from CYP1A2, CYP2B6, and CYP2D6. For the gluc-
uronide conjugate detected, UGT1A9 was found to be the single
largest contributor, with smaller contributions noted from UGT1A3
Kinetic Analysis. The concentration-dependent human microso-
mal metabolism for each of the identified metabolites, M1 through
M8 (excluding M6), was evaluated. Figure 11 shows the graph for
M1, which was found to be representative for the other measured
metabolites except for M2, which is also shown. Under the con-
ditions described above, M6 was not generated in sufficient quan-
tities to conduct kinetic analysis. The relationships between rela-
tive formation rates of metabolites and substrate concentration
showed hyperbolic saturation kinetics for each metabolite. The
Michaelis constants for the oxidative metabolites (M1–M5) and the
dehydrogenated metabolites (M7, M8) extend over a broad range
from 0.97 to 25.2 ?M and 3.8 to 16.5 ?M, respectively (Table 1).
Eadie-Hofstee plots (insets in Fig. 11) for each of the metabolites
were constructed and used in a diagnostic manner to identify
atypical metabolic kinetic behavior (data shown only for M1 and
M2). Metabolites M1, M4, and M5 show monophasic kinetics
consistent with standard hyperbolic Michaelis-Menten kinetics.
The Eadie-Hofstee plots for M2 and M7 exhibited a convex com-
ponent that suggests substrate inhibition. For M3 and M8, the
majority of the data points are consistent with Michaelis-Menten
kinetics; however, there is an indication of autoactivation (sigmoi-
dal response) at the lower concentrations tested. Because authentic
standards for each of the metabolites measured were not available,
absolute determinations for the rate of formation (V) were not
FIG. 9. Negative ion mode product ion spectrum from a neutral loss scan of a
glucuronidated metabolite of 9cUAB30.
FIG. 7. A and B, product ion spectrum of 9cUAB30 in the positive (A) and negative
(B) ion mode.
FIG. 8. A–C, negative ion mode product ion spectra of oxidized metabolite (M4)
(A) and an oxidized plus dehydrogenated metabolite (M5) (B). C, positive ion mode
spectrum of a dehydrogenated metabolite (M8).
IN VITRO METABOLIC CHARACTERIZATION OF 9cUAB30 IN MICROSOMES
This work represents the first investigation of 9cUAB30 metabo-
lism in a P450 enzyme system. Our experimental data show that
9cUAB30 is metabolized to nine phase I oxidative or dehydrogenated
metabolites and one glucuronide conjugate in gender-specific HLM,
RLM, and DLM (Fig. 12). Characterization of the metabolites, deter-
mination of their associated kinetics, and enzyme mapping were
accomplished using LC/MS/MS. Characteristic mass shifts combined
with product ion scans (MS/MS) were used in all the experiments.
Variation in the metabolic profile as a function of gender was only
observed in RLM. This was also the only species observed to produce
M9, identified as a metabolite with two degrees of oxidation. The
presence of M9 as observed in this species at the 2-h time point is
likely a result of excessive oxidation based on the metabolic half-life
of 9cUAB30 in RLM (Fig. 5). This is supported by analysis of the 1-h
time point, which shows the presence of M9 but at a lower relative
concentration (data not shown). The oxidative metabolite M4 was
detected in readily measurable quantities in male RLM yet was barely
detectable above background in the female RLM. No substantial
gender differences were noted in the other species tested; however,
differences in the metabolic profile were noted between species.
Whereas the M1 oxidative metabolite was found to be the most
abundant in RLM, it was among the smallest in HLM and DLM.
Additionally, the ratio of M6/M7 in the RLM was inversely propor-
tional to that found in the HLM. The smallest quantity of these two
metabolites was found in reactions with the DLM, where approxi-
mately equal amounts were measured. The third dehydrogenated
metabolite, M8, was the most abundant in all three species tested. The
presence of three dehydrogenated metabolites each chromatographi-
cally separated and derived from a molecule where only two dehy-
drogenated tetralin ring products are possible may be explained by
cis-trans isomerization of the polyene chain. Conversion between the
cis- and trans-isomeric forms of the parent would allow for more than
the three observed products. Comparable biotransformation for a
structurally related analog, retinoic acid has previously been reported
(Chen and Juchau, 1998). Evidence for P450-catalyzed dehydrogena-
tion of tetralin rings has been shown with testosterone (Nagata et al.,
1986; Korzekwa et al., 1990) and in aliphatic compounds (Rettie et
al., 1995). The presence of the small peak in the ion chromatograms,
which elutes just after 9cUAB30, produced identical mass spectra for
both Q1 mass scans and product ion (MS/MS) scans as compared with
9cUAB30 (data not shown). Mass spectra were also obtained for
authentic standards of cis- and trans-retinoic acid, which showed MS
or MS/MS alone was incapable of differentiating between the two
isomeric forms (data not shown).
Metabolic stability for phase I metabolism exhibited a detectable
difference between genders for both rats and dogs in terms of amount
of substrate remaining after 2 h of incubation. 9cUAB30 was found to
FIG. 10. A–C, enzyme mapping for metabolites M1 through M5
(A), M6 through M8 (B), and UGT (C).
GORMAN ET AL.
be the least stable in RLM, yet exhibited comparable stability in DLM
and HLM. No statistical significant difference was noted between
genders in HLM. The amount of observed glucuronidation for
9cUAB30 was found to be much more extensive compared with
oxidation for all three species tested. Statistically significant differ-
ences in the amount of glucuronidation between genders were noted
for both rats and dogs but not in humans.
Metabolism of xenobiotics can be affected by many parameters,
including genetic polymorphisms, high interindividual variability, and
gender differences. Because variable expressions of individual
isozymes can affect the metabolic fate of a drug candidate, it is
important to determine which P450 enzymes contribute to the meta-
bolic process (Shapiro et al., 1995; Cai et al., 2003). The individual
isozymes responsible for the biotransformation of 9cUAB30 into the
observed metabolites were determined to be primarily in the CYP2C
family with a small contribution from CYP1A2 for the phase I and
from the UGT1A family for glucuronidation based on the groups
tested. The CYP2C family belongs to a group of isozymes that are
involved in metabolizing drugs for which specific isozymes have been
identified (Parkinson, 1996). Surprisingly, there was essentially no
contribution from the CYP3A4 isozyme, which is generally believed
to be an important enzyme in xenobiotic metabolism, as well as being
involved in the oxidation of a broad range of substrates. This enzyme
also typically represents a much higher percentage (20–30%) of the
P450 content in human liver (Shimada et al., 1994). As a result of the
low contribution to the overall metabolism of 9cUAB30, a shared
identity (83%) in amino acid sequence (Aoyama et al., 1989), and
similarity in substrate specificity (Thummel and Wilkinson, 1998),
CYP3A5 was not evaluated. Previous work using human P450
isozymes for both a functional and structurally related compound,
retinoic acid, showed the CYP2C family to be the primary P450
enzymes involved in oxidative metabolism with minor contributions
from CYP3A (Shirley et al., 1996; Nadin and Murray, 1999; McSor-
Michaelis constants and peak area ratios for 9cUAB30 metabolites
Relative Vmaxvalues are represented by the peak area ratio of the metabolite compared with an internal standard.
Metabolites of 9cUAB30
M1 M2M3M4M5M7 M8
0.19 0.140.490.400.04 0.008
3.8 ? 1.8
25.2 ? 6.1 0.97 ? 0.4 9.7 ? 3.215.1 ? 6.39.7 ? 2.616.5 ? 7.8
FIG. 11. Representative kinetic plots for metabolites M1 and M2 from HLM with
Eadie-Hofstee plot inserts. M, metabolite peak area; IS, internal standard peak area;
[S], substrate concentration in micromolars.
FIG. 12. Proposed in vitro metabolic pathways of 9cUAB30.
IN VITRO METABOLIC CHARACTERIZATION OF 9cUAB30 IN MICROSOMES
ley and Daly, 2000). Other work with targretin, an RXR agonist
similar in mechanism of action to 9cUAB30 but different in structure,
has been shown to be metabolized by CYP3A4 to hydrolated ring
products in the tetrahydro-2-naphthalene ring, as well as oxo-deriva-
tives (similar to M5) and glucuronide conjugates (Shirley et al., 1997).
Beyond the 10 isozymes evaluated in the mapping studies, CYP26A
has previously been noted as a highly specific isozyme for metabolism
of retinoic acid (Han and Choi, 1996; Abu-Abed et al., 1998; Sonn-
eveld et al., 1998; Chithalen et al., 2002; Loudig et al., 2005). The
primary contribution of this single isozyme to the metabolism of
retinoic acid is both single and multiple degrees of oxidation. Because
of the commercial unavailability of this isozyme, it was not evaluated
with 9cUAB30; however, it is believed that it would have an active
For glucuronidation, a series of UGT incubated with the substrate
showed UGT1A9 and UGT1A3 as the two main enzymes involved in
glucuronide conjugation. Additionally, a small contribution to gluc-
uronide conjugation was also noted for the UGT1A8 and UGT1A1
enzymes. Previously published data show that glucuronidation of
carboxylic acid-containing compounds is dominated by UGT2B7,
UGT1A3, and UGT1A9 in varying degrees (Sakaguchi et al., 2004).
Additional work has shown that for other classes of compounds
containing a carboxylic acid moiety such as nonsteroidal anti-inflam-
matory drugs, UGT1A3 and UGT1A9 catalyze glucuronidation at
those functional groups (Ebner and Burchell, 1993; Green et al.,
In conclusion, this study has characterized various in vitro meta-
bolic parameters of 9cUAB30 from different species with an emphasis
in humans. Our results indicate that oxidation and glucuronidation are
primary metabolic pathways for 9cUAB30 and that for human sys-
tems these are driven mainly by the CYP2C and UGT1 enzyme
families, respectively. Other conjugation pathways (sulfation, meth-
ylation, acetylation, and glutathione) were not observed to play a role
in the overall metabolism of 9cUAB30. Species-specific in vitro
metabolic profiles showed that the human and dog microsomal sys-
tems were most similar, whereas the rats differed significantly from
both of these. 9cUAB30 was found to have the least amount of
metabolic stability in the rat microsomal system and was found to be
comparable between dog and human systems. Kinetic analysis of the
metabolites indicated that three of the identified metabolites—M1,
M4, and M5—seemed to follow classical hyperbolic kinetics, whereas
M3 and M8 showed evidence of autoactivation, and M2 and M7
showed evidence of substrate inhibition.
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Address correspondence to: Gregory S. Gorman, Southern Research Insti-
tute, 2000 Ninth Avenue South, Birmingham, AL 35205. E-mail: firstname.lastname@example.org
GORMAN ET AL.