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DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 12
Printed in U.S.A.
GLUCURONIDATION OF OPIOIDS, CARBOXYLIC ACID-CONTAINING DRUGS, AND
HYDROXYLATED XENOBIOTICS CATALYZED BY EXPRESSED MONKEY
UDP-GLUCURONOSYLTRANSFERASE 2B9 PROTEIN
MITCHELL D. GREEN, GUY BÉLANGER, DEAN W. HUM, ALAIN BÉLANGER,
AND
THOMAS R. TEPHLY
Department of Pharmacology, The University of Iowa (M.D.G., T.R.T.), and Centre de Recherches en Endocrinologie Moléculaire, Le Centre
Hospitalier de l’Université Laval (G.B., D.W.H., A.B.)
(Received June 19, 1997; accepted August 5, 1997)
ABSTRACT:
derivatives are glucuronidated at higher rates, compared with
oripavines; however, glucuronidation efficiency values (Vmax/KM)
for the compounds are similar. Stably expressed UGT2B9 also
catalyzes the glucuronidation of profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and straight-chain
fatty acids at the carboxylic acid moiety. Monoterpenoid alcohols
and propanolol are glucuronidated at aliphatic hydroxyl positions.
Expressed UGT2B9 exhibits enantioselective glucuronidation for
(R/S)-ibuprofen, (R/S)-propanolol, and (1)/(2)-menthol. The data
suggest that monkey UGT2B9 and human UGT2B7 are functionally
similar.
Glucuronidation is a major conjugation reaction that is catalyzed by
numerous isoforms of UGT.1 These enzymes are localized primarily
in the endoplasmic reticulum and participate in the metabolic elimination of many endogenous compounds and xenobiotics (1). Compounds with a wide variety of chemical moieties, such as amines,
hydroxylated compounds, and carboxylic acids, are substrates for
UGT isoforms. UGTs that are members of the UGT1 gene complex
share common second through fifth exons, with at least 12 separate
first exons coding for proteins with unique amino-terminal domains
(2). In contrast, gene products of the UGT2 family appear to be
transcribed from unique genes (3, 4).
Although much is known about the different UGTs expressed in
rats, rabbits, and humans, less is known about UGTs expressed in
other laboratory animals. Monkeys are commonly used in preclinical
drug metabolism studies, as well as in testing of many compounds,
especially opioid agonists and antagonists, with pharmacological activity in the central nervous system (5–7). Therefore, it is of interest
to characterize UGTs to determine the similarities and differences in
the proteins expressed in monkeys, compared with those expressed in
humans. Recently, a cDNA encoding UGT2B92 has been isolated and
stably expressed in HK293 cells, and the reactivity of the expressed
protein toward endobiotics has been characterized (8). UGT2B9,
isolated from cynomolgus monkey prostate and liver cDNA libraries,
is 89% identical to human UGT2B7 in primary amino acid sequence,
and the two expressed enzymes have been shown to catalyze the
glucuronidation of many common endogenous substrates, such as
androsterone and hyodeoxycholate (8 –11). The major difference in
substrate specificity between expressed human UGT2B7 and monkey
UGT2B9 is the ability of the latter to catalyze the glucuronidation of
17-hydroxylated androgens such as testosterone and dihydrotestosterone (8). Coffman et al. (12) have shown that expressed human
UGT2B7 protein catalyzes the 3-O- and 6-O-glucuronidation of morphine. Jin et al. (11) showed that expressed human UGT2B7 catalyzes
the glucuronidation of many clinically important, carboxylic acidcontaining drugs, such as profen NSAIDs, clofibrate, and valproic
acid. The purpose of this study was to characterize the reactivity of
expressed UGT2B9 with drugs and other xenobiotics. Specifically,
the reactivity of UGT2B9 with opioids and carboxylic acid-containing
drugs was examined.
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UDP-glucuronosyltransferase (UGT) 2B9, isolated from a cynomolgus monkey liver cDNA library, is 89% identical to human UGT2B7
in primary amino acid sequence, and the two expressed enzymes
were previously shown to catalyze the glucuronidation of many
common endogenous substrates. The purpose of the present
study was to characterize the reactivity of expressed UGT2B9 with
important therapeutic agents and other xenobiotics. UGT2B9, stably expressed in human embryonic kidney 293 cells, catalyzes the
3-O- and 6-O-glucuronidation of morphine and the 6-O-glucuronidation of codeine. A number of other morphinan (e.g. naloxone,
naltrexone, and nalorphine) and oripavine (e.g. buprenorphine) derivatives are substrates for this enzyme. In general, morphinan
Materials and Methods
This work was supported by National Institutes of Health Grant GM26221
(T.R.T.), the Medical Research Council of Canada, Fonds de la Recherche en
Santé du Québec (950031–103) (D.W.H.), and Endoresearch.
1
Abbreviations used are: UGT, UDP-glucuronosyltransferase; HK293 cells,
human embryonic kidney 293 cells; NSAIDs, nonsteroidal anti-inflammatory
drugs.
Send reprint requests to: Dr. Thomas R. Tephly, Department of Pharmacology, 2–452 Bowen Science Building, The University of Iowa, Iowa City, IA 52242.
Materials. Aglycone substrates for glucuronidation assays were of the
highest purity available and were purchased from Sigma Chemical Co. (St.
Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI). Geneticin (G418),
saccharolactone, UDP-glucuronic acid, and L-a-phosphatidylcholine (type
XVI-E from egg yolk) were obtained from Sigma. UDP-[U-14C]glucuronic
2
In a previously published abstract (22), this transferase was referred to as
UGT2B18; however, the UGT Nomenclature Committee has since revised some
of the UGT nomenclature and has designated this monkey transferase as
UGT2B9.
1389
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GREEN ET AL.
Results
Glucuronidation of Opioids Catalyzed by UGT2B9. Membrane
preparations derived from HK293 cells stably expressing UGT2B9
protein catalyzed the glucuronidation of morphinan-based and oripavine-based opioids (table 1). Opioid glucuronidation was not found
using membrane preparations from control HK293 cells. Morphine
and hydromorphone were glucuronidated at high rates, whereas naloxone and naltrexone were glucuronidated at lower rates. The observation that codeine was a substrate suggested that expressed UGT2B9
catalyzes 6-O-glucuronidation of morphinan opioids. HPLC analysis
confirmed that UGT2B9 catalyzed the formation of both morphine
3-O- and 6-O-glucuronide, with the rate of 3-O-glucuronide formation
being about 15 times higher than that of 6-O-glucuronide formation.
Removal of the N-methyl group from morphine (normorphine) and
codeine (norcodeine) greatly decreased the rates of conjugation of
these compounds. In general, the rates of glucuronidation for oripavine opioids are lower than those for the morphinan opioids.
Kinetic analysis of opioid glucuronidation catalyzed by expressed
UGT2B9 is shown in table 2. Although morphinan derivatives are
glucuronidated at higher rates, compared with oripavines, the glucuronidation efficiency value (Vmax/KM) of morphine is similar to that of
buprenorphine. The results show that the efficiency of glucuronidation
for nalorphine was highest and that the efficiencies for the other
opioids were comparable. The results also show that the N-alkyl side
chain length appears to be a major influence on the apparent KM.
Compounds with an N-methyl substituent (e.g., hydromorphone, di-
TABLE 1
Glucuronidation of opioids catalyzed by expressed UGT2B9
Assays were performed at 37°C for 0.5–1.0 hr, using membrane
preparations from HK293 cells expressing UGT2B9. The concentration of
UDP-glucuronic acid was 2.0 mM, and all substrates were assayed at pH
8.0. Enzymatic rates are expressed as the mean 6 SD of determinations
made from membrane preparations from at least three different passages of
cells.
Substrate
Glucuronide Formation
pmol/min/mg protein
Morphinan opioids
Morphine
3-Glucuronide
6-Glucuronide
Normorphine
Codeine
Norcodeine
Naloxone
Naltrexone
Nalorphine
Hydromorphone
Dihydromorphine
Oxymorphone
Oripavine opioids
Buprenorphine
Norbuprenorphine
Diprenorphine
270 6 18
18 6 1
2.5 6 0.6
59 6 10
2.6 6 0.5
43 6 9
16 6 4
162 6 15
300 6 53
124 6 44
72 6 5
33 6 5
17 6 6
15 6 6
TABLE 2
Kinetics of glucuronide formation for selected opioids in membrane
preparations from HK293 cells stably expressing UGT2B9 protein
Apparent KM values for the aglycone substrates were determined using
2.0 mM UDP-glucuronic acid. All reactions were conducted at pH 8.0,
except for that with buprenorphine, which was conducted at pH 7.0. Results
are expressed as the mean 6 SE for determinations made using membrane
preparations from three different passages of cultured cells or as the
individual values obtained using membrane preparations from two different
passages of cultured cells. Kinetic values for morphine 6-glucuronide
formation were not determined because of low rates of product formation.
Substrate
Morphine
3-Glucuronide
6-Glucuronide
Codeine
Dihydromorphine
Hydromorphone
Oxymorphone
Naloxone
Nalorphine
Naltrexone
Buprenorphine
a
Apparent KM
Vmax
mM
pmol/min 3 mg
330 6 14
NDa
239, 156
980 6 70
1390 6 280
702, 775
45, 48
60, 65
50, 67
41, 40
302 6 11
ND
82, 61
390 6 28
570 6 125
237, 235
35, 61
310, 280
19, 24
46, 34
Vmax/KM
(3100)
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acid (225 mCi/mmol) was purchased from ICN Radiochemicals (Costa Mesa,
CA). Protein assay reagents were obtained from Bio-Rad (Richmond, CA).
Stable Expression of Monkey UGT2B9 Protein. The establishment and
characterization of an HK293 cell line stably expressing UGT2B9 protein were
reported previously (8). The HK293 cells expressing UGT2B9 were grown in
Dulbecco’s modified Eagle’s medium containing 4.5 mM glucose, 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10% fetal bovine serum,
and 700 mg/ml geneticin, in a humidified incubator with an atmosphere of 5%
CO2, at 37°C. Membrane preparations were made by differential ultracentrifugation using a modification of the method of Battaglia et al. (13), as described
by King et al. (14). Glucuronidation activities for the substrates used in the
present studies were not detected in nontransfected HK293 cell homogenates.
UGT Assays. Membrane pellets from HK293 cells expressing UGT2B9
protein were suspended in 10 mM Tris-buffered saline (pH 7.4), containing 0.5
mM dithiothreitol, and were gently resuspended using a Potter-Elvehjem
homogenizer. Opioid glucuronidation was determined using the method described by Puig and Tephly (15). The HPLC method of Svensson et al. (16)
was used to separate morphine 3-O- and 6-O-glucuronides, and the corresponding fractions were collected and quantitated using scintillation counting.
Glucuronidation activities toward all other aglycone substrates were analyzed
using the TLC method described by Bansal and Gessner (17), as modified (18).
All assay mixtures (0.1-ml final volume) contained 50 mM Tris-HCl, 10 mM
MgCl2, 100 mg/ml phosphatidylcholine, 8.5 mM saccharolactone, 2.0 mM
UDP-glucuronic acid (0.25 mCi/assay), and 0.5 mM aglycone substrate, unless
otherwise indicated. Reactions in which the aglycone substrate was omitted
served as controls, and the minimal counts obtained were subtracted from
values for reactions where aglycone was present. Glucuronidation assays were
conducted using pH 7.5 buffer, unless otherwise indicated. All enzymatic
assays were conducted at 37°C under conditions that produced linear product
formation with respect to time (0.5–2 hr) and protein concentration (up to 75
mg/0.1-ml assay).
Apparent KM values for aglycone substrates were estimated by varying the
aglycone concentration from approximately one-fifth to five times the apparent
KM values, using a fixed concentration (2.0 mM) of UDP-glucuronic acid.
Kinetic values were determined using assay conditions yielding linear product
formation with respect to protein concentration and reaction times. Kinetic
parameters were calculated using the program Enzyme Kinetics (Trinity Software, Plymouth, NH).
92
37
40
41
32
103
475
37
99
ND, not determined.
hydromorphine, and morphine) have higher apparent KM values than
do compounds with longer N-alkyl substitutions, such as naloxone,
nalorphine, and buprenorphine.
Glucuronidation of Carboxylic Acid-Containing Drugs. A number of clinically important drugs were tested to determine whether
they were substrates for expressed UGT2B9. Several carboxylic acid-
SUBSTRATE SPECIFICITY FOR UGT2B9
1391
TABLE 3
Glucuronidation of drugs catalyzed by expressed UGT2B9
Assays were performed at 37°C for 0.5–1.0 hr, using membrane
preparations from HK293 cells expressing UGT2B9. The concentration of
UDP-glucuronic acid was 2.0 mM, aglycone concentrations were 0.5 mM,
and all substrates were assayed at pH 7.5. Enzymatic rates are expressed as
the mean 6 SD of determinations made from membrane preparations from
at least three different passages of cells or as the average of two
determinations.
Glucuronide Formation
(S)-Ibuprofen
(R)-Ibuprofen
Ketoprofen
Fenoprofen
Naproxen
Clofibrate
Ciprofibrate
Valproic acid
Meclofenamic acid
Furosemide
Ethacrynic acid
Diflunisal
(R)-Propanolol
(S)-Propanolol
Oxazepam
Chloramphenicol
pmol/min/mg protein
161 6 7
44 6 4
55
34 6 4
16 6 2
2
40
7
13
15
5
12
21 6 2
12 6 1
4
3
containing NSAIDs were substrates for expressed UGT2B9 protein
(table 3). Of the profen NSAIDs tested, S-ibuprofen exhibited the
highest glucuronidation rate. Of the two fibrate hypolipidemic agents
tested, the glucuronidation rate for ciprofibrate was higher than that
for clofibrate. Other carboxylic acid-containing drugs, such as valproic acid, furosemide, and diflunisal, were also substrates for expressed UGT2B9. Interestingly, expressed UGT2B9 also catalyzed
the glucuronidation of straight-chain saturated fatty acids (fig. 1). Of
the fatty acids tested, n-decanoic acid was the best substrate. Fatty
acids with longer or shorter alkyl chains were glucuronidated at lower
rates than decanoic acid. Short-chain carboxylic acids with aromatic
substitutions one or two carbons removed from the carboxylic acid
moiety (2-naphthylacetic acid, triphenylacetic acid, and 3,3,3-triphenylpropionic acid) were glucuronidated at low rates (12, 6, and 10
pmol/min/mg protein, respectively).
Stably expressed UGT2B9 also catalyzed the glucuronidation of
drugs and xenobiotics with aliphatic hydroxyl groups. Propanolol,
oxazepam (table 3), and monoterpenoid alcohols (table 4) are weak
substrates for the expressed protein. Of the monoterpenoid alcohols
tested, borneol had the highest glucuronidation rate for expressed
UGT2B9. Indeed, of all the compounds tested in this study, borneol
exhibited the highest glucuronidation rate.
Of the other drugs tested, measurable glucuronidation activity was
not detected for acetaminophen, probenecid, phenolphthalein,
hexafluoro-2-propanol, and the 4-hydroxy metabolite of tamoxifen.
The reactivity of expressed UGT2B9 toward coumarins and flavonoids was also examined. 4-Methylumbelliferone was glucuronidated at a moderate rate (48 pmol/min/mg protein), but glucuronidation of umbelliferone, scopoletin, esculetin, and 4hydroxycoumarin was not detected. Likewise, glucuronidation of 7hydroxyflavone, apigenin, naringenin, chrysin, and galangin by
UGT2B9 was not detected. The only flavonoids found to be substrates
for expressed UGT2B9 were fisetin (111 pmol/min/mg), quercetin
(142 pmol/min/mg), and genistein (18 pmol/min/mg). Sapogenins,
such as tigogenin and hecogenin, were not substrates for UGT2B9.
FIG. 1. Glucuronidation of n-alkyl fatty acids by expressed UGT2B9.
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Substrate
Glucuronidation of straight-chain fatty acids catalyzed by expressed
UGT2B9 was conducted using pH 6.4 bis-Tris (f) or pH 7.5 Tris (F) buffer.
Glucuronidation rates are given in picomoles of glucuronide formed per
minute per milligram of protein. The results represent the mean values obtained using three different membrane preparations, and the SDs are contained
within the data points.
Kinetic Analysis of the Glucuronidation of Xenobiotics by Expressed UGT2B9. Apparent enantioselectivity was observed for the
glucuronidation of (R/S)-ibuprofen, (R/S)-propanolol, and (1)/(2)menthol, but not for (R/S)-b-citronellol (tables 3 and 4). Kinetic
analysis was conducted for some of these compounds to determine
whether the differences in glucuronidation rates for the enantiomers
were due to different affinities of the compounds for the expressed
enzyme. The results of these studies are shown in table 5. No significant differences were found in the apparent KM values for (R/S)ibuprofen, (R/S)-propanolol, and (1)/(2)-menthol, suggesting that the
stereoisomers interact with the expressed protein in similar manners.
The higher glucuronidation rates observed for (S)-ibuprofen, (R)propanolol, and (2)-menthol, compared with their enantiomers, can
be explained entirely by their higher Vmax values.
pH Effects on the Glucuronidation of Opioids and Carboxylic
Acids Catalyzed by Expressed UGT2B9. Opioids and carboxylic
acids represent very diverse classes of substrates when their pKa
values are considered. Glucuronidation of the basic opioids by expressed UGT2B9 membrane preparations exhibited three types of pH
dependence. The pH curves for morphine (fig. 2A), hydromorphone,
and codeine were similar, in that glucuronidation rates increased with
increasing pH up to about pH 7.7 and then plateaued. In contrast, the
glucuronidation rates for buprenorphine (fig. 2B) and norbuprenorphine peaked at pH 7.0 and then declined. The glucuronidation of
naltrexone (fig. 2C) and naloxone exhibited very little pH dependence
in the range of pH 6.7– 8.4. These results suggest that, for morphinan
opioids, the ionization potential of the N-methyl substituent contributes to the binding of the substrate to the active site of the enzyme.
Longer N-alkyl substitutions are more likely to be unionized over the
range of pH values used in the present study.
The glucuronidation of carboxylic acids catalyzed by expressed
UGT2B9 was also found to be pH dependent. Whereas pH 7.4 was
found to be optimum for the glucuronidation of the ibuprofen enantiomers, naproxen (fig. 2D) and the intermediate-chain fatty acids
were glucuronidated at higher rates at low pH (about pH 6.4). Kinetic
analysis of naproxen glucuronidation showed that at pH 6.4 the
apparent KM was lower and the Vmax was higher than the values
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GREEN ET AL.
TABLE 4
Glucuronidation of monoterpenoid alcohols catalyzed by expressed UGT2B9
Assays were performed at 37°C for 0.5–1.0 hr, using membrane
preparations from HK293 cells expressing UGT2B9. The concentration of
UDP-glucuronic acid was 2.0 mM, aglycone concentrations were 0.5 mM,
and all substrates were assayed at pH 7.5. Enzymatic rates are expressed as
the mean 6 SD of determinations made from membrane preparations from
at least three different passages of cells or as the average of two
determinations.
Glucuronide Formation
Borneol
(1)-Menthol
(2)-Menthol
Thymol
(1)-Isomenthol
(1)-Neomenthol
(6)-Isoborneol
endo-Norborneol
(R) 2 (1)-b-Citronellol
(S) 2 (2)-b-Citronellol
(2)-Carveol
pmol/min/mg protein
546 6 40
101 6 4
265 6 15
14
206
17
183
12
127
135
85
TABLE 5
Kinetics of glucuronide formation for selected xenobiotics in membrane
preparations from HK293 cells stably expressing UGT2B9 protein
Apparent KM values for the aglycone substrates were determined at pH
7.5, unless otherwise noted, using 2.0 mM UDP-glucuronic acid. Results are
expressed as the individual values obtained using membrane preparations
from different passages of cultured cells.
Substrate
Naproxen
pH 7.5
pH 6.5
(R)-Ibuprofen
(S)-Ibuprofen
(R)-Propanolol
(S)-Propanolol
(1)-Menthol
(2)-Menthol
Apparent KM
Vmax
mM
pmol/min 3 mg
368, 403
56, 62
117, 59
133, 54
390, 420
440, 375
225, 148
140, 161
26,
40,
53,
218,
44,
18,
129,
372,
24
41
45
176
50
19
112
303
Vmax/KM
(3100)
7,
71,
45,
164,
11,
4,
57,
266,
6
66
76
326
12
5
76
188
obtained at pH 7.5 (table 5). This resulted in an approximately 10-fold
increase in the glucuronidation efficiency at pH 6.4. These results
suggest that the unionized forms of carboxylic acids are the preferred
forms of the substrates.
Discussion
UGT2B9 is a simian enzyme whose cDNA has been recently
isolated from liver and prostate and expressed in HK293 cells, to
investigate the substrate specificity of the encoded protein. Bélanger
et al. (8) showed that UGT2B9 mRNA is expressed in liver and in
steroid target tissues such as ovary, epididymis, testis, and prostate
and that the expressed enzyme demonstrated a wide substrate specificity for C18, C19, and C21 steroids and bile acids. The major
difference in substrate specificity between expressed human UGT2B7
and monkey UGT2B9 is the ability of the latter to catalyze the
glucuronidation of 17-hydroxylated androgens, such as testosterone
and dihydrotestosterone. Among the C18 steroids glucuronidated by
FIG. 2. pH optima for the glucuronidation of selected substrates.
The glucuronidation pH optima for morphine (A), buprenorphine (B), naltrexone (C), and naproxen (D) were determined using bis-Tris (M) or Tris (E)
buffers, as described in Materials and Methods.
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Substrate
expressed UGT2B9 are the catechol estrogens 4-hydroxyestrone and
4-hydroxyestradiol. Recent studies in our laboratory have suggested
that human UGTs that catalyze the glucuronidation of 4-hydroxylated
catechol estrogens also catalyze the glucuronidation of morphine (19).
In the present study, we found that expressed UGT2B9 catalyzes the
glucuronidation of opioids at the aromatic 3- and aliphatic 6-hydroxyl
positions, that of profen NSAIDs, fibrate hypolipidemic agents, and
straight-chain fatty acids at the carboxylic acid moiety, and that of
monoterpenoid alcohols at aliphatic hydroxyl groups.
In general, the rates of glucuronidation for oripavine opioids are
lower than those for morphinan opioids, suggesting that these compounds may be poorer substrates for the enzyme. However, kinetic
analysis showed that the glucuronidation efficiency values (Vmax/KM)
of the compounds are similar. The rate of morphine 6-O-glucuronide
formation catalyzed by expressed UGT2B9 (6%), compared with the
rate of morphine 3-O-glucuronide formation, is similar to the comparable value found by Rane et al. (20) for rhesus monkey liver microsomes (4%). Using expressed human UGT2B7, the rate of morphine
6-O-glucuronide formation was found to be about 13% of the rate of
morphine 3-O-glucuronide formation (12), and the ratio obtained was
similar to that observed in human liver microsomes. Because the
morphine 6-O-glucuronidation rate was low for expressed monkey
UGT2B9, it was not possible to accurately determine the apparent KM
for morphine for the 6-O-glucuronidation reaction. However, for
human morphine glucuronidation catalyzed by UGT2B7, the apparent
KM for morphine for 6-O-glucuronide formation is similar to that for
3-O-glucuronidation (12).
Morphinan-based opioid glucuronidation catalyzed by expressed
UGT2B9 demonstrates opioid structure-activity relationships similar
to those found for expressed rat UGT2B1, except that rat UGT2B1
does not catalyze morphine 6-O-glucuronide formation (14). Sanchez
1393
SUBSTRATE SPECIFICITY FOR UGT2B9
Glucuronidation of naproxen at pH 6.5 results in a lower apparent
KM for the substrate and a higher Vmax, compared with assays conducted at pH 7.5. This resulted in a 10-fold increase in the glucuronidation efficiency for naproxen at pH 6.5. It is unclear which
values are more physiologically relevant, because the microenvironment at the active site of UGTs is not known. Therefore, pH dependence for UGT substrates must be determined to identify the appropriate kinetic values.
Simian UGT2B9 and human UGT2B7 are 89% identical in primary
amino acid sequence, and expressed UGT2B9 was previously shown
to catalyze the glucuronidation of many C18, C19, and C20 steroids and
bile acids (8). The substrate specificity of monkey UGT2B9 for
endogenous compounds and xenobiotics is clearly different from
those observed for human UGT2B4 (10), UGT2B15 (18), and
UGT2B17 (24). Of the human UGTs that have been characterized to
date, UGT2B9 appears to be most similar to UGT2B7, based on
substrate specificity of the expressed enzymes for endogenous compounds and xenobiotics.
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et al. (21) showed that the presence and length of the N-alkyl side
chain of opioids affect the ability of the compounds to interact with
the UGT opioid binding site. They found that N-demethylated opioids
were poor inhibitors of morphine glucuronidation in rat and rabbit
liver microsomes. In contrast, they found that opioids with longer
N-alkyl side chains had low Ki values for the inhibition of morphine
glucuronidation. In the present study, we have shown that normorphine and norcodeine are glucuronidated at very low rates (1/100 and
1/25, respectively), compared with their N-methylated analogues. The
very low glucuronidation rates for these compounds made it impossible to accurately determine the kinetic values for these compounds,
but the data suggest that the demethylated compounds are poor
substrates. Saturation of the 7,8-double bond of the morphinan ring
structure resulted in compounds (i.e. dihydromorphine and hydromorphone) that exhibited much higher apparent KM values and similar or
somewhat higher Vmax values, compared with morphine. Oxymorphone, naloxone, and naltrexone differ only in their N-alkyl substituents (N-methyl, N-allyl, and N-cyclopropylmethyl, respectively). As
predicted by the results of Sanchez et al. (21), naloxone and naltrexone have apparent KM values 1 order of magnitude lower than that for
the N-methylated oxymorphone. Similar results have recently been
observed for expressed human UGT2B7 (12, 22).
Expressed monkey UGT2B9 catalyzes the stereoselective glucuronidation of menthol, ibuprofen, and propanolol. Similarly to the
results reported using expressed human UGT2B7 (11), expressed
monkey UGT2B9 preferentially glucuronidated the S-enantiomer of
ibuprofen. The S/R ratio of 3.7/1 obtained using standard assay
conditions was similar to the Vmax S/R ratio of 4/1 obtained using
optimal kinetic assay conditions. Likewise, the (2)/(1)-menthol glucuronidation ratio of 2.6/1 under standard assay conditions was the
same as the Vmax (2)/(1)-menthol glucuronidation ratio of 2.8/1. In
contrast, the Vmax S/R glucuronidation ratio for propanolol of 1/2.6
was somewhat different from the ratio of 1/1.7 obtained under standard assay conditions. These results are probably explained by the fact
that for the propanolol standard glucuronidation assays the substrate
concentration (0.5 mM) is only slightly higher that the apparent KM
values for (S)- and (R)-propanolol (about 0.4 mM), whereas for
ibuprofen and menthol the standard assay substrate concentration is
4 –5 times the apparent KM values. Although large differences are
observed in the Vmax values of the enantiomers studied, the apparent
KM values are not different. These results suggest that the stereoisomers interact similarly with the enzyme but that the energetics of the
reactions are different.
In addition to the carboxylic acid-containing profen NSAIDs and
fibrates, expressed simian UGT2B9 catalyzes the glucuronidation of
fatty acids. Fatty acids have also been shown to be substrates for rat
UGT2B1, an enzyme that also catalyzes opioid glucuronidation (23).
Similarly to the results of Pritchard et al. (23) using rat UGT2B1,
UGT2B9 glucuronidates n-decanoic acid at higher rates, compared
with compounds with shorter and longer side chains. The glucuronidation of fatty acids by other UGTs has not been investigated. Caprylic,
decanoic, and dodecanoic acids were glucuronidated at higher rates at
pH 6.4, compared with pH 7.5, suggesting that the unionized carboxylic group is the preferred substrate. The disappearance of pH dependence for the glucuronidation of fatty acids with side chains longer
than decanoic acid is probably due to the increasing lipophilicity of
these compounds. With highly lipophilic fatty acids, glucuronidation
may be less pH dependent. Short-chain fatty acids are quite water
soluble, and the lower pH may be necessary for a significant amount
of the compound to be in the unionized, and therefore more lipophilic,
form.
Acknowledgments. We thank Martin Beaulieu and Eric Lévesque
for isolation of the UGT2B9 cDNA.
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