Download n-glucuronidation of some 4-arylalkyl-1h

0090-9556/02/3003-295–300$3.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
DMD 30:295–300, 2002
Vol. 30, No. 3
477/966313
Printed in U.S.A.
N-GLUCURONIDATION OF SOME 4-ARYLALKYL-1H-IMIDAZOLES BY RAT, DOG,
AND HUMAN LIVER MICROSOMES
SANNA KAIVOSAARI, JARMO S. SALONEN,
AND
JYRKI TASKINEN
Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, Finland (S.K., J.T.); and Orion Pharma, Preclinical and
Clinical Research and Development, Turku, Finland (J.S.S.)
(Received June 7, 2001; accepted December 5, 2001)
This article is available online at http://dmd.aspetjournals.org
ABSTRACT:
levomedetomidine exhibited a 60-fold Vmax/Km value compared with
dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was
formed from levomedetomidine. Dog liver microsomes formed Nglucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity,
with apparent Vmax values ranging from 0.29 to 0.73 nmol/min/mg of
protein and apparent Km values from 279 to 1640 ␮M. Rat liver microsomes glucuronidated these compounds at a barely detectable
rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for
4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not
explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.
Glucuronidation is a phase II conjugation reaction catalyzed by a
family of UDP-glucuronosyltransferase isoenzymes (UGTs1; EC
2.4.1.17) (Clarke and Burchell, 1994). Compounds possessing a nucleophilic O or N atom in their structure are the most common
substrates for UGTs. Glucuronidation to a hydroxyl group (usually
formed in a phase I reaction) is probably the best-characterized phase
II reaction of drug metabolism. In contrast, N-glucuronidation represents a less examined pathway. Various nitrogen-containing functional groups, which are extremely common in drug chemistry, are
susceptible to direct glucuronidation without any phase I modification. Compounds that form N-glucuronides include aliphatic and
aromatic amines and various heterocycles. N-Glucuronidation constitutes a major detoxification reaction in the metabolism of some drugs
and other xenobiotics (Hawes, 1998); for example, 63% of the oral
dose of lamotrigine is excreted in human urine as a quaternary
triazine-linked N-glucuronide (Cohen et al., 1987; Sinz and Remmel,
1991). Significant, substrate-dependent differences between species in
their ability to form N-glucuronides have been observed (Chiu and
Huskey, 1998).
The new drug discovery paradigm emphasizing early prediction of
absorption-distribution-metabolism-excretion properties has created a
great interest in understanding the metabolism potential of different
chemical structures. The imidazole ring is one of the common structural motifs in drug molecules susceptible to direct glucuronidation.
Imidazole N-glucuronide was detected as a metabolite of croconazole
isolated from rabbit urine and as a major metabolite of tioconazole
isolated from human urine (Takeuchi et al., 1989; Macrae et al.,
1990). These glucuronides, in addition to glucuronides of nafimidone
alcohol and imiloxan isolated from human urine (Rush et al., 1990,
1992), are representatives of N⫹-glucuronides (i.e., quaternary ammonium glucuronides carrying a permanent positive charge). Quaternary N⫹-glucuronides are formed from imidazole compounds, which
carry the original substituent in one of the imidazole ring nitrogens. In
contrast, tertiary N-glucuronides are formed from N-unsubstituted
imidazoles [e.g., from methylbiphenyl-C4-imidazole (Huskey et al.,
1994)].
Human UGT isoform(s) responsible for the glucuronidation of
imidazoles have not yet been identified. Several isoforms of the
UGT1A subfamily, including 1A3, 1A4, 1A6, 1A9 (Orzechowski et
al., 1994; Green and Tephly, 1996, 1998; Green et al., 1998), and also
This work was presented in part at the Drug Metabolism Workshop and
International Society for the Study of Xenobiotics Meeting, June 11–16, 2000, St.
Andrews, Scotland. The abstract was published in Drug Metab Rev 32:26.
1
Abbreviations used are: UGT, UDP-glucuronosyltransferase; UDPGA, UDPglucuronic acid; MPV-207 A IV, 4-(2,6-dimethylphenyl)methyl-1H-imidazole;
MPV-295 A IV, 4-(2,6-dimethylphenyl)ethyl-1H-imidazole; HPLC, high-performance liquid chromatography; tR, total retention time in HPLC.
Address correspondence to: Sanna Kaivosaari, Department of Pharmacy,
Division of Pharmaceutical Chemistry, P.O. Box 56, FIN-00014 University of
Helsinki, Finland.
295
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 24, 2016
N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both
enantiomers of medetomidine, detomidine, atipamezole, and two
other closely related compounds) by rat, dog, and human liver
microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes
formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high
activity, with apparent Vmax values ranging from 0.59 to 1.89 nmol/
min/mg of protein. In comparison, apparent Vmax values for two
model compounds forming the N-glucuronides 4-aminobiphenyl
and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent Km
value of 4.0 ␮M and a high specificity constant (Vmax/Km) of 256
compared with 4-aminobiphenyl (Km, 265 ␮M; Vmax/Km, 19) and
amitriptyline (Km, 728 ␮M; Vmax/Km, 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes;
296
KAIVOSAARI ET AL.
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 24, 2016
UGT2B7 (Stevens et al., 2001), have been reported to be involved in
N-glucuronidation of xenobiotics in humans.
4-Arylalkyl-1H-imidazoles belong to a class of pharmacologically
active C4-imidazole compounds, which may form tertiary imidazole
N-glucuronides. The pharmacological effect of these drugs is based on
agonism of both pre- and postsynaptic ␣2-receptors (MacDonald and
Virtanen, 1992). Two of these compounds, detomidine and medetomidine (Fig. 1), are therapeutically used as analgesic sedatives for
animals. Dexmedetomidine (Precedex; Abbott Laboratories, Abbott
Park, IL) was recently approved for clinical use for sedation of
patients in intensive care. Biotransformation is the most important
pathway in the elimination of these compounds (Salonen et al., 1988,
1991; Salonen and Eloranta, 1990). When the metabolism of levomedetomidine was first studied in human liver microsomes, direct glucuronidation to the imidazole ring nitrogen was observed (Lehtonen
and Salonen, 1997). According to preliminary results, N-glucuronidation is an important route in the metabolism of dexmedetomidine in
humans in vivo.
In our study, species differences in N-glucuronidation of detomidine, both enantiomers of medetomidine, atipamezole, and some other
structurally related 4-arylalkyl-1H-imidazoles, between rat, dog, and
human liver microsomes were determined. The effects of minor
differences in the chemical structures of these compounds on their
glucuronidation rates were observed. UGT activities for these 4-arylalkyl-1H-imidazoles were elucidated also in expressed human UGT
isoforms 1A3, 1A4, 1A6, and 1A9.
Materials and Methods
Chemicals. Saccharolactone, 4-nitrophenol, amitriptyline, nortriptyline,
4-aminobiphenyl, and scopoletin were purchased from Sigma (St. Louis, MO).
UDPGA was obtained from Roche Molecular Biochemicals (Mannheim, Germany) and [14C]UDPGA from PerkinElmer Life Sciences (Boston, MA).
4-Arylalkyl-1H-imidazoles levomedetomidine [(⫺)-R-medetomidine], dexmedetomidine [(⫹)-S-medetomidine], detomidine, atipamezole, MPV-207 A IV,
MPV-295 A IV, and [3H]levomedetomidine (Fig. 1) were kindly provided by
Orion Pharma (Espoo, Finland). Entacapone was also provided by Orion
Pharma. All other reagents were purchased from commonly used suppliers and
were of the highest grade available.
Assays for Liver Microsomal UGTs. All reaction mixtures contained 50
mM phosphate buffer, pH 7.4, 10 mM MgCl2, 5 mM saccharolactone, and
depending on the aglycon, 0.5 to 10,000 ␮M substrate in a total volume of 100
␮l. 4-Nitrophenol, scopoletin, entacapone, and 4-aminobiphenyl were added in
4 ␮l of dimethyl sulfoxide and amitriptyline in 5 ␮l of methanol to increase the
solubility of these substrates. For each substrate and enzyme preparation, an
optimal concentration [0 – 0.05 mg/ml corresponding to 0 –1 (mg/mg) detergent/protein ratio] of Triton X-100 was used as a detergent. Pooled human liver
microsomes were purchased from Human Biologics (Scottsdale, AZ). Dog
liver microsomes from a male beagle were prepared as previously described
(Salonen, 1991), and pooled rat liver microsomes from six male Wistar rats
were prepared as described in Luukkanen et al. (1997). Microsomes were
added at a protein concentration of 0.02 to 0.3 mg/ml. Preincubation was
carried out at 37°C for 10 min, and incubation at 37°C for 10 to 60 min was
initiated by the addition of 5 mM UDPGA. A blank sample was prepared and
incubated in the same way, but substrate or UDPGA was replaced with
incubation buffer. All reactions were shown to be linear with respect to protein
concentration and incubation time. Reactions were terminated with 100 ␮l of
ice-cold methanol (4-aminobiphenyl) or with 10 ␮l of 4 M perchloric acid
(other substrates) while maintaining the reaction mixture in an ice bath.
Reaction mixtures were centrifuged at 14,000 rpm for 5 min, and 80 ␮l of the
supernatant was injected into the HPLC column.
Assays for Expressed Human UGT Isoenzymes. All reaction mixtures
contained 50 mM phosphate buffer, pH 7.4, 10 mM MgCl2, 5 mM saccharolactone, and 500 ␮M substrate in a total volume of 100 ␮l. Human UGT1A3
expressed in baculovirus-infected insect SF-9 cells was purchased from Panvera (Madison, WI), and UGT1A4 expressed in human B-lymphoblastoid
FIG. 1. Chemical structures of substrates.
Chiral center marked with 多.
AHH-1 cells was purchased from GENTEST (Woburn, MA). Human
UGT1A6 and UGT1A9 were expressed in Chinese hamster lung fibroblast
V79 cells using Semliki Forest virus vector, as previously described (Forsman
et al., 2000). A protein concentration of 0.02 to 2 mg/ml and an incubation time
of 45 min were used in conditions in which less than 5% of substrate was
1.7
106.0
1.67
N.D.
N.D.
5.07 ⫾ 0.40
0.56
N.D.
265 ⫾ 35
728
19.0
0.8
19 ⫾ 1
0.52 ⫾ 0.03
N.D.
N.D.
28.0
963
⫾3.9
66.6
628⫾15
726.0
195 ⫾ 4
269 ⫾ 27
30.6 ⫾ 2.3a
290 ⫾ 16a
106.0
N.D.
N.D.
N.D.
0.001b
N.D.
0.004b
1.9
0.3
0.4
0.5
0.4
1.4
0.54 ⫾ 0.03
0.29 ⫾ 0.04
0.39 ⫾ 0.04
0.48 ⫾ 0.03
0.69 ⫾ 0.08
0.73
279 ⫾ 24
1150 ⫾ 140
985 ⫾ 195
1050 ⫾ 80
1640 ⫾ 300
503
117.0
2.0
16.0
256.0
48.0
19.0
1.89a
0.74
1.22 ⫾ 0.03
1.02 ⫾ 0.10
1.66 ⫾ 0.06
0.59 ⫾ 0.02
16a
371
78 ⫾ 13
4.0 ⫾ 0.9
34 ⫾ 1
31 ⫾ 2
4-Arylalkyl-1H-imidazoles
Levomedetomidine
Dexmedetomidine
Detomidine
Atipamezole
MPV-207 A IV
MPV-295 A IV
Phenolic compounds
4-Nitrophenol
Amines
4-Aminobiphenyl
Amitriptyline
Nortriptyline
Vmax/Km
␮l/min/mg protein
Vmax
nmol/min/mg protein
Km
␮M
␮l/min/mg protein
Vmax/Km
Vmax
nmol/min/mg protein
␮M
Km
Vmax/Km
Vmax
nmol/min/mg protein
Km
␮M
␮l/min/mg protein
Rat Liver Microsomes
Dog Liver Microsomes
Human Liver Microsomes
Substrate
TABLE 1
Kinetics of glucuronidation in rat, dog, and human liver microsomes
All parameters represent an average ⫾ S.D. of three to four replicate series or an average of two replicate series using a minimum of seven substrate concentration levels. Samples were prepared as described under Materials and Methods.
Results
Kinetics of Glucuronidation in Human Liver Microsomes. All
studied 4-arylalkyl-1H-imidazoles were substrates for human liver
microsomal UGTs, with apparent Vmax values ranging from 0.59 to
1.89 nmol/min/mg of protein (Table 1). These capacities were comparable with the Vmax values measured for the model primary and
tertiary amine substrates of N-glucuronidation (i.e., 4-aminobiphenyl
and amitriptyline). The secondary amine nortriptyline, which is the
demethylated metabolite of amitriptyline, was not glucuronidated at a
detectable level. 4-Nitrophenol, the model phenolic compound forming an O-glucuronide, was glucuronidated at clearly the highest capacity (Vmax, 30.6 nmol/min/mg of protein).
In general, 4-arylalkyl-1H-imidazoles were approximately 10-fold
higher affinity substrates than 4-nitrophenol, 4-aminobiphenyl, and
amitriptyline. Atipamezole showed the highest affinity for human
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 24, 2016
consumed. Preincubation was carried out at 37°C for 10 min, and the incubation at 37°C was initiated by the addition of 5 mM UDPGA. A blank sample
was prepared and incubated in the same way, but the substrate or UDPGA was
replaced with incubation buffer. Reactions were terminated with 100 ␮l of
ice-cold methanol (4-aminobiphenyl) or with 10 ␮l of 4 M perchloric acid
(other substrates) while maintaining the reaction mixture in an ice bath.
Reaction mixtures were centrifuged at 14,000 rpm for 5 min, and 80 ␮l of the
supernatant was injected into the HPLC column.
HPLC Conditions. Glucuronides of various substrates were analyzed on a
model 1100 or 1090 HPLC instrument (Hewlett Packard, Waldbronn, Germany), as described earlier (Kaivosaari et al., 2001). Briefly, glucuronides were
separated on a Symmetry 150 ⫻ 3.9-mm C18 column (Waters, Milford, MA)
or a Hypersil BDS 250 ⫻ 4-mm C18 column (Hewlett Packard). The mobile
phase consisted of 50 mM phosphate buffer, pH 3.0, and methanol, with the
exception of acid-labile 4-aminobiphenyl glucuronide, which was analyzed by
an application of the method by Babu et al. (1996) using a mixture of 20 mM
phosphate buffer, pH 6.8, and methanol. Glucuronides were detected by a
model 1100 UV detector (Hewlett Packard).
Glucuronide Quantitation Using [14C]UDPGA. Since reference N-glucuronides of the studied compounds were not available as quantitation standards
for UV, radioactivity detection was used for quantitation of the formed
N-glucuronide products. Separate quantitation samples were prepared to quantify glucuronide conjugates of each substrate, as described earlier (Kaivosaari
et al., 2001). Briefly, quantitation was performed by adding [14C]UDPGA
(0.1– 0.4 ␮Ci) and a variable amount (2.5–500 ␮M) of unlabeled UDPGA to
the UGT incubation mixture. A substrate concentration of 500 ␮M and a
protein concentration and incubation time of up to 0.5 mg/ml and 60 min,
respectively were used. Glucuronide products were quantified using a model
9701 radioactivity detector (Reeve Analytical, Glasgow, UK) equipped with a
heterogeneous 200-␮l flow cell packed with silanized cerium-activated lithium
glass (GS1/TSX; Reeve Analytical) positioned after a model 1100 UV detector
(Hewlett Packard). A standard curve was attained for the UV detector in which
the peak area of the glucuronide on the UV detector (mAU⫻s) was presented
as a function of the quantified amount of glucuronide (picomoles) attained
from the radioactivity detector. This curve was used to quantify glucuronide
products formed in UGT assay samples containing 5 mM UDPGA but no
14
C-labeled UDPGA.
Glucuronide Quantitation Using [3H]Levomedetomidine. Levomedetomidine glucuronidation in human liver microsomes was determined by adding
[3H]levomedetomidine (0.5–1.5 ␮Ci) to the UGT assay mixture. Glucuronide
conjugates were quantified by a model 150TR radioactivity monitor (Packard,
Meriden, CT) equipped with a homogeneous flow cell (500 ␮l), using Monoflow 3 scintillation fluid (National Diagnostics, Atlanta, GA) at a flow rate of
3 ml/min.
Calculation of the Apparent Kinetic Parameters. To determine glucuronidation kinetics, UGT activities were measured at a minimum of seven
substrate concentrations with two to four replicates at each concentration level.
Apparent kinetic parameters Km and Vmax were estimated using a nonlinear
least-square fit to the Michaelis-Menten equation by the Leonora enzyme
kinetics program version 1.0 by Cornish-Bowden (1995).
N.D., indicates that glucuronide formation was not detectable (detection limit 0.8 pmol/min/mg of protein).
a
These results were published in Kaivosaari et al., 2001.
b
UGT activity at 500 ␮M substrate concentration rather than Vmax. Glucuronidation assays of 4-arylalkyl-1H-imidazoles by rat liver microsomes (n ⫽ 2) were conducted at 37°C for 60 min with 500 ␮M substrate, 1.59 mg/ml microsomal protein, and
0.05 mg/ml Triton X-100.
297
N-GLUCURONIDATION OF 4-ARYLALKYL-1H-IMIDAZOLES
298
KAIVOSAARI ET AL.
14
C radiochromatograms (model 9701; Reeve Analytical) of UGT assays of medetomidine enantiomers.
A, two glucuronides were formed from dexmedetomidine in human liver microsomes with a tR/s of 7.7 and 9.7 min (unreacted [14C]UDPGA tR, 1.8 min); B, only one
glucuronide (tR, 7.7 min) was formed from dexmedetomidine in dog liver microsomes. C, human liver microsomes formed only one glucuronide (tR, 7.1 min) from
levomedetomidine, the other enantiomer of medetomidine. UGT assays were conducted as described under Materials and Methods using 0.5 mg/ml microsomal protein,
2000 ␮M dexmedetomidine, or 500 ␮M levomedetomidine and 50 ␮M (0.2 ␮Ci) UDPGA.
liver microsomal UGTs, with a very low apparent Km of 4.0 ␮M. The
glucuronidation rate of atipamezole decreased when substrate concentration exceeded 25 ␮M, evidently due to substrate inhibition. This
type of inhibition was not observed at substrate concentrations below
1000 ␮M for other 4-arylalkyl-1H-imidazoles.
Atipamezole was the best substrate also in terms of the Vmax/Km
ratio, which describes the efficiency of the enzymatic reaction, with a
ratio of 256. Differences in the Vmax/Km ratios between 4-arylalkyl1H-imidazoles were striking, ranging from 256 (atipamezole) to 2.0
(dexmedetomidine). Although Vmax/Km ratios of similar magnitude
were attained for the best 4-arylalkyl-1H-imidazole substrates and
4-nitrophenol, this was a result of 4-arylalkyl-1H-imidazoles being
higher affinity (lower Km) yet lower capacity (lower Vmax) substrates
compared with 4-nitrophenol.
Some notable differences were apparent in the glucuronidation
rates of various 4-arylalkyl-1H-imidazoles by human liver microsomes despite the very similar chemical structures of these compounds. First, compared with the other enantiomer levomedetomidine,
dexmedetomidine was a poor substrate, with 23-fold higher Km value
and 2.6-fold lower Vmax value. Two distinct glucuronides with different retention times (7.7 and 9.7 min) in HPLC were formed from
dexmedetomidine (Fig. 2a), representing 70 and 30% of the total
glucuronidation at 2000 ␮M substrate concentration, respectively.
The apparent kinetic parameters were determined only for the glucuronide eluting at a tR value of 7.7 min. In contrast, only one
glucuronide conjugate (tR, 7.1 min) was formed from levomedetomidine (Fig. 2c).
Second, detomidine, a homolog of medetomidine, showed 4.9-fold
lower affinity than levomedetomidine, although the Vmax values were
of similar magnitude. MPV-207 A IV and MPV-295 A IV, differing
structurally by just one methylene group, showed very similar Km
values. Yet, a 2.8-fold difference in Vmax in favor of the compound
with a shorter alkyl chain (MPV-207 A IV) was observed.
Kinetics of Glucuronidation in Dog Liver Microsomes. All studied 4-arylalkyl-1H-imidazoles were substrates for dog liver microsomal UGTs, with apparent Vmax values ranging from 0.29 to 0.73
nmol/min/mg of protein (Table 1). The glucuronidation capacity of
4-aminobiphenyl (Vmax, 0.52 nmol/min/mg of protein) was compara-
ble to that of 4-arylalkyl-1H-imidazoles. Amitriptyline and nortriptyline were not glucuronidated at detectable levels. 4-Nitrophenol was
glucuronidated at a very high capacity, with Vmax being ⬎250-fold
compared with the substrates forming N-glucuronides.
In general, N-glucuronidation of 4-arylalkyl-1H-imidazoles in dog
liver microsomes occurred at a lower affinity than in human liver
microsomes, with apparent Km values ranging from 279 to 1640 ␮M.
Reliable estimation of Km and Vmax values was difficult because for
an unknown reason glucuronidation rates decreased when millimolar
substrate concentrations were reached. The solubility of 4-arylalkyl1H-imidazoles prevented the use of ⬎5 mM substrate concentrations,
but glucuronidation rates decreased even before solubility limits were
exceeded, at ⬎1 mM concentrations. When all data points, including
the inhibition part of the curve, were included in the calculation of
kinetic parameters and the “substrate inhibition equation” was used,
the data points did not fit into the equation. Thus, the kinetic parameters given in Table 1 were obtained excluding the data points in the
inhibition part of the plot and using the “conventional nonlinear
Michaelis-Menten equation” of the Leonora enzyme kinetics program.
4-Arylalkyl-1H-imidazoles, of which levomedetomidine showed the
highest Vmax/Km ratio of 1.9, showed considerably lower efficiency for
dog liver microsomal UGTs compared with 4-nitrophenol and 4-aminobiphenyl, with Vmax/Km ratios of 725 and 27, respectively.
Glucuronidation of medetomidine was enantioselective also in dog
liver microsomes, with the Vmax/Km value of levomedetomidine being
8-fold higher compared with dexmedetomidine. Unlike with human
liver microsomes, only one glucuronide product of dexmedetomidine
(, 7.7 min) was detected (Fig. 2b). Similarly, only one glucuronide
conjugate (tR, 7.1 min) was formed from levomedetomidine.
Kinetics of Glucuronidation in Rat Liver Microsomes. Rat liver
microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a
very low rate (Table 1); only MPV-295 A IV and atipamezole glucuronides were formed at a detectable level. Amitriptyline and nortriptyline were not glucuronidated at detectable levels. The only
substrate forming an N-glucuronide at a sufficient rate to determine
kinetic parameters was 4-aminobiphenyl, but it was not a very good
substrate compared with 4-nitrophenol, which was a 61-fold better
substrate in terms of Vmax/Km ratio.
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 24, 2016
FIG. 2.
299
N-GLUCURONIDATION OF 4-ARYLALKYL-1H-IMIDAZOLES
TABLE 2
Glucuronidation activity in expressed human UGT isoenzymes
UGT assays were conducted as described under Materials and Methods using 500 ␮M
substrate concentration. All values represent an average of two determinations.
UGT1A3
UGT1A4
UGT1A6
UGT1A9
nmol/min/mg protein
4-Arylalkyl-1H-imidazoles
Levomedetomidine
Detomidine
Atipamezole
Phenolic compounds
4-Nitrophenol
Scopoletin
Entacapone
Amines
4-Aminobiphenyl
N.D.
N.D.
N.D.
0.009
0.014
0.007
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.M.
0.052
N.M.
N.M.
N.M.
N.M.
5.87
N.M.
N.M.
N.M.
N.M.
4.22
0.002
0.225
N.D.
0.031
N.D., indicates that glucuronide formation was not detected (detection limit 0.8 pmol/min/mg
of protein); N.M., not measured.
Detergent Activation of Liver Microsomal UGTs. Triton X-100
was added to the incubation mixture to fully activate liver microsomal
UGTs. The optimal detergent concentration was determined separately for each enzyme source and substrate before determining the
kinetic parameters. Optimal Triton X-100 concentrations varied between 0 to 0.05 mg/ml, corresponding to Triton/protein ratios (mg/
mg) between 0 to 1 (data not shown). Generally, glucuronidation of
4-arylalkyl-1H-imidazoles in human liver microsomes was strongly
activated, up to 5-fold, by the detergent (Fig. 3a). In contrast, Triton
X-100 had no or only a minor effect on the glucuronidation of
4-arylalkyl-1H-imidazoles in dog liver microsomes (Fig. 3b).
Glucuronidation Activity in Expressed Human UGT Isoenzymes. Scopoletin, 4-aminobiphenyl, 4-nitrophenol, and entacapone
were used as model substrates to measure glucuronidation activity in
expressed human UGT1A3, UGT1A4, UGT1A6, and UGT1A9 isoenzymes, respectively (Table 2). The 4-arylalkyl-1H-imidazoles studied
(levomedetomidine, atipamezole, and detomidine) were not substrates
for human UGT1A3, UGT1A6, or UGT1A9. Human UGT1A4
formed N-glucuronides of these 4-arylalkyl-1H-imidazoles, but the
glucuronidation rates were low, below 0.015 nmol/min/mg of protein.
Discussion
Human liver microsomes glucuronidated 4-arylalkyl-1H-imidazoles at a very high efficiency; the Vmax/Km ratios (e.g., for atipamezole and levomedetomidine) were 256 and 117, respectively, com-
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 24, 2016
FIG. 3. Triton X-100 strongly activated glucuronidation of MPV-207 A IV in (A)
human liver microsomes (protein concentration, 0.1 mg/ml; [S], 100 ␮M) but
had a minor effect on (B) dog liver microsomes (protein concentration, 0.2 mg/
ml; [S], 500 ␮M).
pared with a Vmax/Km ratio of 106 for 4-nitrophenol, a well
characterized UGT substrate forming an O-glucuronide (Table 1).
From the high efficiencies and high affinities (low Km), it can be
predicted that N-glucuronidation is a major metabolic route in the
elimination of these 4-arylalkyl-1H-imidazoles in humans. This is an
important finding because these compounds are glucuronidated without
any prior phase I metabolism. Preliminary in vivo studies with dexmedetomidine support the importance of this metabolic route in humans.
Dog liver microsomes also formed glucuronides of 4-arylalkyl-1Himidazoles. The Vmax/Km ratios were 0.5 and 1.9 for atipamezole and
levomedetomidine, respectively, compared with 726 for 4-nitrophenol
(Table 1).
Rat liver microsomes formed glucuronides of 4-arylalkyl-1H-imidazoles at a very low rate; only atipamezole and MPV-295 A IV
glucuronides were formed at detectable levels. Our results with 4-arylalkyl-1H-imidazoles, which are C4-imidazole compounds, are in
good agreement with the findings of Huskey et al. (1993, 1994), who
studied species differences in N-glucuronidation of methylbiphenyltetrazole, -triazole, and -imidazole compounds. They concluded
that human UGT(s) responsible for N-glucuronidation preferentially
conjugated methylbiphenyl-C4-imidazole (and methylbiphenyl-1,2,4triazole), whereas rat UGTs glucuronidated methylbiphenyl-C4-imidazole at a very low rate.
Among 4-arylalkyl-1H-imidazoles, atipamezole showed the highest
affinity for human liver microsomal UGTs, with an exceptionally low
apparent Km of 4.0 ␮M, and levomedetomidine showed the highest
capacity, with an apparent Vmax of 1.89 nmol/min/mg of protein
(Table 1). Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at 5- to 100-fold lower affinity (higher Km) than
human liver microsomes. Levomedetomidine was the best substrate
with a Km of 279 ␮M, and MPV-207 AIV showed a Vmax of 0.73
nmol/min/mg of protein (Table 1). At high substrate concentrations
(⬎1 mM), glucuronidation of 4-arylalkyl-1H-imidazoles and 4-aminobiphenyl in dog liver microsomes showed substrate inhibition,
making the reliable estimation of kinetic parameters difficult.
In principle, either of the imidazole ring nitrogens (Fig. 1) of the
studied 4-arylalkyl-1H-imidazoles could be glucuronidated. However,
N-glucuronidation of these compounds seemed to be regiospecific,
and only a single glucuronide product of each substrate was observed,
with one exception (dexmedetomidine). Huskey et al. (1994) discovered in their study using NMR spectroscopy that in human liver
microsomes the favored N-glucuronidation site in C4-methylbiphenyl
imidazole was the nitrogen located two bonds away from the substituted carbon (i.e., N1). The nitrogen located next to the substituted
carbon (i.e., N3 was glucuronidated at a lower rate). Moreover,
300
KAIVOSAARI ET AL.
somes, but the dog also formed glucuronide conjugates of these compounds, albeit at a lower affinity. The rat formed N-glucuronides of
4-arylalkyl-1H-imidazoles at a very low rate. Further studies are needed
to reveal which human UGT isoenzymes, in addition to UGT1A4, are
responsible for the N-glucuronidation of these compounds.
References
Babu SR, Lakshmi VM, Huang GP-W, Zenser TV, and Davis BB (1996) Glucuronide conjugates
of 4-aminobiphenyl and its N-hydroxy metabolites. Biochem Pharmacol 51:1679 –1685.
Bansal SK and Gessner T (1980) A unified method for the assay of uridine diphosphoglucuronyltransferase activities toward various aglycones using uridine diphospho[U-14C]glucuronic
acid. Anal Biochem 109:321–329.
Chiu S-HL and Huskey S-EW (1998) 1996 ASPET N-glucuronidation of xenobiotics symposium: species differences in N-glucuronidation. Drug Metab Dispos 26:838 – 847.
Clarke DJ and Burchell B (1994) The uridine diphosphate glucuronosyltransferase multigene
family: function and regulation, in Conjugation-Deconjugation Reactions in Drug Metabolism
and Toxicity (Kauffman FC ed) pp 3– 43, Springer-Verlag, Berlin, Germany.
Cohen AF, Land GS, Breimer DD, Yuen WC, Winton C, and Peck AW (1987) Lamotrigine, a
new anticonvulsant. Pharmacokinetics in normal humans. Clin Pharmacol Ther 42:535–541.
Cornish-Bowden A (1995) in Fundamentals of Enzyme Kinetics (Cornish-Bowden A ed) Portland Press, London, UK.
Forsman T, Lautala P, Lundström K, Monastyrskaia K, Ouzzine M, Burchell B, Taskinen J, and
Ulmanen I (2000) Production of human UDP-glucuronosyltransferases 1A6 and 1A9 using the
Semliki Forest virus expression system. Life Sci 67:2473–2484.
Green MD, King CD, Mojarrabi B, Mackenzie PI, and Tephly TR (1998) Glucuronidation of
amines and other xenobiotics catalyzed by expressed human UDP-glucuronosyl transferase
1A3. Drug Metab Dispos 26:507–512.
Green MD and Tephly TR (1996) Glucuronidation of amines and hydroxylated xenobiotics and
endobiotics catalyzed by expressed human UGT1.4 protein. Drug Metab Dispos 24:356 –362.
Green MD and Tephly TR (1998) 1996 ASPET N-glucuronidation of xenobiotics symposium:
glucuronidation of amine substrates by purified and expressed UDP-glucuronosyltransferase
proteins. Drug Metab Dispos 26:860 – 867.
Hawes EM (1998) 1996 ASPET N-glucuronidation of xenobiotics symposium: N ⫹ glucuronidation, a common pathway in human metabolism of drugs with a tertiary amine
group. Drug Metab Dispos 26:830 – 837.
Huskey S-EW, Doss GA, Miller RR, Schoen WR, and Chiu S-HL (1994) N-glucuronidation
reactions II. Relative N-glucuronidation reactivity of methylbiphenyl tetrazole, methylbiphenyl triazole, and methylbiphenyl imidazole in rat, monkey, and human hepatic microsomes.
Drug Metab Dispos 22:651– 658.
Huskey S-EW, Miller RR, and Chiu S-HL (1993) N-glucuronidation reactions. I. Tetrazole
N-glucuronidation of selected angiotensin II receptor antagonists in hepatic microsomes from
rats, dogs, monkeys, and humans. Drug Metab Dispos 21:792–799.
Kaivosaari S, Salonen JS, Mortensen J, and Taskinen J (2001) High-performance liquid chromatographic method combining radiochemical and ultraviolet detection for determination of
low activities of uridine 5⬘-diphosphate-glucuronosyltransferase. Anal Biochem 292:178 –187.
Lehtonen T and Salonen JS (1997) Göteborg 6th European ISSX meeting 1997: Nglucuronidation of levomedetomidine in vitro is catalyzed by human but not by rat liver
microsomes (Abstact). ISSX Proc 11:208.
Lett E, Kriszt W, de Sandro V, Ducrotoy G, and Richert L (1992) Optimal detergent activation
of rat liver microsomal UDP-glucuronosyl transferases toward morphine and 1-naphthol:
contribution to induction and latency studies. Biochem Pharmacol 43:1649 –1653.
Liu Z and Franklin MR (1984) Separation of four glucuronides in a single sample by highpressure liquid chromatography and its use in the determination of UDP glucuronosyltransferase activity toward four aglycones. Anal Biochem 142:340 –346.
Luukkanen L, Elovaara E, Lautala P, Taskinen J, and Vainio H (1997) Characterization of
1-hydroxypyrene as a novel marker substrate of 3-methylcholanthrene-inducible phenol UDPglucuronosyltransferase(s). Pharmacol Toxicol 80:152–158.
MacDonald E and Virtanen R (1992) Review of the pharmacology of medetomidine and
detomidine: two chemically similar ␣2 adrenoceptor receptor agonists used as veterinary
sedatives, in Animal Pain (Short CE and Van Poznak A eds) pp 181–191, Churchill Livingstone, New York.
Macrae PV, Kinns M, Pullen FS, and Tarbit MH (1990) Characterization of a quaternary,
N-glucuronide metabolite of the imidazole antifungal, tioconazole. Drug Metab Dispos 18:
1100 –1102.
Orzechowski A, Schrenk D, Bock-Hennig BS, and Bock KW (1994) Glucuronidation of
carcinogenic arylamines and their N-hydroxy derivatives by rat and human phenol UDPglucuronosyltransferase of the UGT1 gene complex. Carcinogenesis 15:1549 –1553.
Rush WR, Alexander OF, Hall DJ, Dow RJ, Tokes L, Kurz L, and Graham DJM (1990) The
metabolism of nafimidone hydrochloride in the dog, primates and man. Xenobiotica 20:123–132.
Rush WR, Hall DJ, Graham DJM, and Selby IA (1992) The metabolism of imiloxan hydrochloride in healthy male volunteers. Xenobiotica 22:237–246.
Salonen JS (1991) Tissue-specificity of hydroxylation and N-methylation of arylalkylimidazoles.
Pharmacol Toxicol 69:1– 4.
Salonen JS and Eloranta M (1990) Biotransformation of medetomidine in the rat. Xenobiotica
20:471– 480.
Salonen JS, Vuorilehto L, Eloranta M, and Karjalainen A (1988) Metabolism of detomidine in
the rat II. Characterization of metabolites in urine. Eur J Drug Metab Pharmacokinet
13:59 – 65.
Sinz MW and Remmel RP (1991) Isolation and characterization of a novel quaternary ammonium-linked glucuronide of lamotrigine. Drug Metab Dispos 19:149 –153.
Stevens JC, Fayer JL, and Cassidy KC (2001) Characterization of 2-[[4-[[2-(1H-tetrazol-5ylmethyl)phenyl]methoxy]phenoxy]methyl] quinoline N-glucuronidation by in vitro and in
vivo approaches. Drug Metab Dispos 29:289 –295.
Takeuchi M, Nakano M, Mizojiri K, Iwatani K, Nakagawa Y, Kikuchi J, and Terui Y (1989)
Quaternary ammonium glucuronide of croconazole in rabbits. Xenobiotica 19:1327–1336.
Winsnes A (1969) Studies on the activation in vitro of glucuronyltransferase. Biochim Biophys
Acta 191:279 –291.
Downloaded from dmd.aspetjournals.org at ASPET Journals on October 24, 2016
C2-methylbiphenyl imidazole, in which both nitrogens are immediate
neighbors of the substituted carbon, was not glucuronidated. The
4-arylalkyl-1H-imidazoles in our study are C4-substituted imidazoles;
therefore, it is possible that their glucuronidation occurs preferentially
in the N1 nitrogen (Fig. 1). A plausible explanation for this is that the
orientation leading to N3-glucuronidation is sterically hindered, resulting in high Km binding in the active site of the enzyme. The
formation of both N1- and N3-glucuronides of these 4-arylalkyl-1Himidazoles cannot, however, be excluded since the C18 column used
in our HPLC system may have not separated them. Confirming the
exact position of glucuronidation would require NMR spectroscopy.
The glucuronidation of medetomidine by human liver microsomes was
highly stereospecific; levomedetomidine showed a 60-fold apparent specificity constant Vmax/Km (which gives the relative rates of the reactions in
an equimolar mixture) compared with dexmedetomidine. The lower
efficiency of dexmedetomidine glucuronidation was mainly due to higher
a Km value; dexmedetomidine showed the lowest binding affinity of all
4-arylalkyl-1H-imidazoles. Dexmedetomidine produced two N-glucuronide products when it was incubated with human liver microsomes (Fig.
2), whereas all the other 4-arylalkyl-1H-imidazoles produced only one.
The lack of regioselectivity in the case of dexmedetomidine glucuronidation may be explained by comparable (low) affinity for both binding
modes (N1 and N3). The regioselectivity of N-glucuronidation for all the
other 4-arylalkyl-1H-imidazoles may be caused by one high-affinity (N1)
and one low-affinity (N3) binding mode, leading to the detection of only
one glucuronide product (N1).
Triton X-100 was used as a detergent to activate liver microsomal
UGTs of the three species. If too low of a detergent concentration is
used, the active sites of UGTs located in the lumen of the endoplasmic
reticulum are not fully revealed, whereas excessively high concentrations tend to denature the protein. Therefore, an optimal detergent
concentration was determined separately for each microsome preparation and substrate before kinetic characterization. The optimal Triton X-100 concentration for 4-arylalkyl-1H-imidazoles to activate
liver microsomal UGTs of the three species varied between 0 to 0.05
mg/ml (Triton/protein ratio (mg/mg) 0 – 0.5), which was in good
agreement with previous studies, the optimum normally being between 0.2 to 1 Triton/protein ratio (Winsnes, 1969; Bansal and Gessner, 1980; Liu and Franklin, 1984; Lett et al., 1992).
The human UGT1A subfamily isoforms reported to catalyze Nglucuronidation are UGT1A3, UGT1A4, UGT1A6, and UGT1A9
(Orzechowski et al., 1994; Green and Tephly, 1996; Green et al.,
1998). All of these isoenzymes were tested in our study for their
ability to glucuronidate 4-arylalkyl-1H-imidazoles. Only UGT1A4
formed glucuronides of these compounds at a low rate, between 0.007
and 0.014 nmol/min/mg of protein (Table 2). Glucuronidation activity
of human UGT1A4 for 4-aminobiphenyl was 25-fold higher compared with levomedetomidine, yet the difference was only 2.7-fold
when Vmax values of these compounds were compared in human liver
microsomes. Therefore, it seems likely that in addition to UGT1A4,
some other UGT isoenzyme(s) contribute to the glucuronidation of
4-arylalkyl-1H-imidazoles in human liver. Detectable amounts of
glucuronides were not formed from 4-arylalkyl-1H-imidazoles by
human expressed UGT1A3, UGT1A6, or UGT1A9. However, the
involvement of UGT1A3 in the glucuronidation of 4-arylalkyl-1Himidazoles cannot be excluded since the activity for scopoletin in the
insect cell-expressed UGT1A3 used in our study was very low compared with a previous study on UGT1A3 (Green et al., 1998).
In conclusion, N-glucuronidation of several structurally related 4-arylalkyl-1H-imidazoles was elucidated by rat, dog, and human liver microsomes. Significant species differences were observed; glucuronidation
occurred at high rate and at the highest affinity in human liver micro-