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Department of Entomology and Cancer Research Center, University of California, Davis, California
(Received November 4, 2002; accepted March 20, 2003)
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-carboxylate; however, the rodent tissue appeared to
perform this transformation more rapidly. After a 60-min incubation in rat
hepatic microsomes, the percentage of residual CDU, the -carboxylate,
and the intermediary -hydroxyl were about 20%, 20%, and 50%,
respectively. Carbon monooxide inhibited the metabolism of CDU by rat hepatic
microsomes, suggesting that the initial step is catalyzed by cytochrome P450.
Further metabolism was enhanced by the addition of NAD, suggesting that
dehydrogenases are associated with intermediate metabolic steps. Regardless,
the ultimate product of microsomal metabolism,
12-(3-cyclohexyl-ureido)-dodecanoic acid, is also an excellent sEH inhibitor
with several hundred-fold higher solubility, supporting the hypothesis that
CDU has prodrug characteristics. These findings will facilitate the rational
design and optimization of sEH inhibitors with better physical properties and
improved metabolic stability.
). These enzymes are widely distributed throughout the animal
and plant kingdoms and not only metabolize epoxides of drugs and xenobiotics,
but also catalyze the hydration of endogenous compounds. In mammals, the
microsomal EH and soluble EH (sEH), which detoxify mutagenic, carcinogenic,
and xenobiotic epoxides (Wixtrom et al., 1985), have broad and complementary
substrate selectivities (Hammock et al.,
1997). The sEH rapidly hydrates fatty acid epoxides
(Gill and Hammock, 1979) and
seems especially involved in the metabolism of epoxides of arachidonic acid
(Chacos et al., 1983;
Halarnkar et al., 1989;
Zeldin et al., 1995) and
linoleic acid (Moghaddam et al.,
1997; Zheng et al.,
2001). Epoxides of arachidonic acid (epoxyeicosatrienoic acids)
are endogenous regulators (Capdevila et
al., 2000) that influence blood pressure by modulating cardiac
output, vascular resistance, renal fluid, and electrolyte balance, whereas the
diols of linoleate epoxides have been implicated in inflammatory disorders,
such as acute respiratory distress syndrome
(Moghaddam et al., 1997), and
may be endogenous regulators of vascular permeability and inflammation
(Slim et al., 2001).
Therefore, the modulation of endogenous lipid epoxides using sEH inhibitors
may have therapeutic benefits in both hypertensive and inflammatory
conditions.
The development of potent and stable inhibitors of sEH has facilitated the
inhibition of this enzyme in both in vitro and in vivo models
(Morisseau et al., 1999;
Yu et al., 2000;
Fang et al., 2001). Oral
availability is also an important factor for long-term in vivo studies.
However, the poor solubility and relatively short duration of action limits
the use of the most potent urea-based sEH inhibitors reported to date. We have
reported that some of our potent inhibitors are metabolized very quickly in
hepatic microsomes (Watanabe and Hammock,
2001). Although the low inhibitor solubility can be understood by
log P calculations, the absorption, distribution, metabolism, and excretion
properties of these chemicals most likely account for their short time of
action. Therefore, understanding the route of metabolism should provide
information leading to the design of more stable inhibitors. In addition, we
hypothesize that metabolic transformations of the aliphatic -terminal
of one class of inhibitory lead compounds will not alter their inhibition
potency but, rather, will enhance their water solubility. To test this
hypothesis, we have focused the current study on the metabolism of
1-cyclohexyl-3-dodecyl-urea (CDU), one potent inhibitor of sEH
(Morisseau et al., 2002). To
assess the metabolism of these compounds, a specific and sensitive analytical
method is required. High-performance liquid chromatography with tandem mass
spectrometry (LC/MS-MS) provides a useful tool for drug metabolism studies.
Here we report the development of a LC/MS-MS-based method for the simultaneous
determination of CDU and its major metabolites, and the use of this method to
investigate the microsomal metabolism of this sEH inhibitor. The obtained
results are used to propose refinements in the optimal structure of sEH
inhibitors for in vivo applications.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
ß-Nicotinamide adenine dinucleotide phosphate sodium salt (NADP),
ß-nicotinamide adenine dinucleotide (NAD), glucose-6-phosphate
dehydrogenase, D-glucose 6-phosphate monosodium salt, and
aminobenzotriazole were obtained from Sigma-Aldrich (St. Louis, MO), and
anhydrous magnesium chloride was from Aldrich Chemical Co. (Milwaukee, WI).
HPLC-grade methanol, acetonitrile, and ethyl acetate, reagent-grade monobasic
monohydrate sodium phosphate, anhydrous dibasic sodium phosphate, and formic
acid (88%) were purchased from Fisher Scientific (Pittsburgh, PA). Water
(>18.0 M
) used was purified by the NANO pure II system (Barnstead,
Newton, MA).
LC/MS-MS. The LC/MS-MS experiments were carried out using a
Micromass Quattro Ultima triple quadrupole tandem mass spectrometer
(Micromass, Manchester, UK) equipped with atmospheric pressure ionization
source [atmospheric z-spray pressure chemical ionization or electrospray
ionization (ESI) interface]. The HPLC system consisted of a Waters model 2790
separations module (Waters, Milford, MA) equipped with a Waters model 2487
dual 
absorbance detector. The mass spectrometer was coupled to the
outlet of the HPLC column or syringe pump for optimization with PEEK tubing.
Data were manipulated with MassLynx software (Ver. 3.5).
An XTerra MS C18 column (30 x 2.1 mm i.d., 3.5 µm; Waters) was used with a flow rate of 0.3 ml/min at ambient temperature. Solvents A and B were 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. Solvents were degassed by vacuum. Mobile phases were mixed with a linear gradient from 40% B to 100% B over 0 to 5 min, and then isocratic for 8 min with 100% B. The post-run was carried out to equilibrate the column to the initial conditions for 1 min before the next run. Five microliters of standard and the extracted microsome samples were injected onto the column.
The ESI was performed in the positive ion mode with a capillary voltage at 1.0 kV. The source and the desolvation temperature were set at 100°C and 300°C, respectively. Cone gas (N2) and desolvation gas (N2) were maintained at flow rates of 130 and 630 l/h, respectively. The optimum cone voltages were set at 80 V for CDU, 80 to 85 V for metabolites, and 100 V for CTU (internal standard), respectively. Mass spectra of the precursor ions were obtained by syringe pump infusions, while scanning over the range of 50 to 350 m/z at 3 s/scan. Data were acquired in the multichannel analysis mode and continuum mode. Quantitative analysis was performed in the multiple reaction monitoring (MRM) mode with a dwell time of 0.6 s. Ultra pure argon (99.9999%) was used as a collision gas at a pressure of 2.5 milli-torr for collision-induced dissociation.
Sample Preparation. CDU, CUDA, and CTU solutions (50–200 µg/ml) were prepared as stock solutions by dissolving the pure compounds in methanol. Standard solutions were stored at 4°C in the dark before use. Standard solutions (1 µg/ml) were prepared for MS optimization study. The concentration range in the calibration studies varied from 2 to 500 ng/ml. A stock solution of CTU for use as an internal standard was prepared in methanol and diluted to a concentration of 500 ng/ml with methanol.
Microsomal Incubation. Ten male Fisher 344 rats (8 weeks old;
Charles River Laboratories, Inc., Wilmington, MA) and 5 male Fisher 344 rats
(8 weeks old; Charles River) treated with 1.5 mmol/kg clofibrate were used for
the preparation of pooled liver microsomes
(Guengerich, 1982). Protein
concentration and -hydroxylation of lauric acid were determined by the
Bradford method (Bradford,
1976) and a radiometric method with [14C]lauric acid as
substrate (Orton and Parker,
1982). A pooled male human liver (n = 10) microsome
preparation (Gentest, Woburn, MA) was also used to evaluate CDU metabolism,
allowing a comparison with rodent data.
For CDU metabolism studies, 0.05 mg of liver microsomal protein (890 µl) were brought to a final volume of 1 ml in 100 mM sodium phosphate buffer at pH 7.4. This protein was preincubated for 5 min in open glass tubes immersed in a shaking bath at a constant temperature of 37°C. After this preincubation, 10 µl of a 100 µM solution of CDU in methanol was added, and the reaction was initiated by the addition of 100 µl of the NADPH generating system. The NADPH regenerating system consisted of 2 mM NADP, 57 mM glucose 6-phosphate, 3.5 units/ml glucose-6-phosphate dehydrogenase, and 50 mM magnesium chloride dissolved in 100 mM sodium phosphate buffer at pH 7.4. Each incubation mixture (1 ml total volume) was incubated in a shaking water bath kept at 37°C for 5, 10, 20, 30, 40, or 60 min. A control was prepared by the addition of 1 ml of ethyl acetate after adding the NADPH generating system. Reactions were terminated by the addition of 1 ml of cold ethyl acetate, and kept in ice water until isolation. A 200-µl aliquot of 500 ng/ml CTU was added to the samples. The samples were then vortexed and centrifuged at 6000 rpm (4000 g) for 5 min. The aqueous phases were extracted twice with ethyl acetate, extracts were combined and dried under nitrogen. The residue was reconstituted in 1 ml of methanol. Aliquots (5 µl) were injected onto the LC-MS-MS system.
To investigate the metabolic pathways involved in CDU transformation,
cofactors, inhibitors, and microsomes from either clofibrate-fed or control
rats were selectively employed. Specifically, NADPH and carbon monoxide were
used to evaluate P450 metabolism, while NAD was used as a dehydrogenase
cofactor. Clofibric acid is a known inducer of CYP4A and ß-oxidation
(Gibson et al., 1982).
Moreover, ABT was used to inhibit CYP4A. After a 10-min incubation of CDU with
microsomes and the NADPH generating system, carbon monooxide gas was bubbled
in the mixture to inhibit NADPH-dependent P450 metabolism. Samples were
incubated for an additional 50 min, followed by extraction and LC/MS-MS
analysis as described above. Finally 600 µl of additional microsomes and
100 µl of 2 mM NAD were added to 300-µl aliquots of microsomes after 40
min of incubation; then, microsomes were incubated for 40 min at 37°C.
After termination by the addition of ethyl acetate, metabolites in microsomes
were also analyzed by LC/-MS-MS.
sEH Activity Assay. Dissociation constants were determined by
following the method described by Dixon
(1972) for competitive
tight-binding inhibitors, using
[3H]1,3-diphenyl-trans-propene oxide as substrate
(Borhan et al., 1995).
Inhibitors at concentrations between 10 and 100 nM were incubated in
triplicate for 5 min in sodium phosphate buffer, pH 7.4, at 30°C with the
recombinant enzyme (2 nM sEH) (Grant et
al., 1993). Substrate (10 ≤ [S]final ≤ 50 µM)
then was added. Velocity was measured as described
(Borhan et al., 1995).
Log P Measurement. CDU or CUDA (10 mg) was added to 4 ml of 1-octanol in a 10-ml glass centrifuge tube, and samples were sonicated for 5 min. Four milliliters of 100 mM phosphate buffer (pH 7.4) then was added to the sample solutions. Then sample solutions were incubated in a shaking bath at 37°C for 24 h. After centrifugation at 6000 rpm for 5 min, 1 ml of the water layer was used for further extraction. Two hundred microliters of 500 ng/ml CTU was added to the samples, and samples were extracted twice with 800 µl of ethyl acetate. Extracts were combined and dried under nitrogen. The residue was reconstituted in 1 ml of methanol. Aliquots (5 µl) were injected onto the LC/MS-MS system.
Kinetics of Metabolism. Linear conditions for the formation of hydroxylate metabolite were evaluated regarding protein content and incubation time for rat hepatic microsomes. The optimum conditions for the kinetics study were 0.05 mg/ml protein concentration of microsomes at 37°C for a 2-min incubation. A substrate concentration range of 100 to 3000 nM was used for the kinetic study. Kinetic data (Km and Vmax) were analyzed by Michaelis-Menten and Hanes-Woolf plots. The intrinsic clearance was calculated using the following equation: v Clint = Vmax/Km.
Statistical Analysis. Analysis of statistical significance, where appropriate, was performed using Student's t test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Analysis Optimizations for CDU and Its Metabolites. The metabolism of CDU in rat hepatic microsomes was investigated using tandem mass spectrometry with a 13-min HPLC solvent gradient. CDU was efficiently ionized in the positive mode due to the presence of nitrogens in the structure. To optimize ionization and fragmentation conditions, 1 µg/ml CDU was delivered into the mass spectrometer using a syringe pump with a 10 µl/min flow rate. The protonated molecule m/z 311 (parent ion) [M + H]+ was the most abundant ion, and the acetonitrile adduct ion was also observed in the Q1 scan spectrum with ESI. For MS-MS experiments, samples were separated chromatographically, and protonated ions for each of the analytes were found as follows: CDU, m/z 311; CHDU, m/z 327; CODU, m/z 325; and CUDA, m/z 341, as shown in Table 1. The ESI tandem mass product ion spectra resulting from collision-induced dissociation of each molecular ion are depicted in Fig. 1. As expected, similarities in the fragment patterns (N-C bond cleavage) were observed for all analytes. As shown in Table 1, the most abundant product ions were m/z 229 (CDU), m/z 202 (CHDU), m/z 200 (CODU), m/z 198 (CUDA), and m/z 257 (CTU; internal standard), respectively. Therefore, these product ions were used for the MRM scan. To optimize MS-MS conditions, the collision voltages between 10 and 32 eV were evaluated. Considering sensitivity, background, and characterization of analyte, optimum collision voltages were set for each target (Table 1).
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Chromatographic resolution of the CDU metabolites was accomplished with a 30-mm reversed phase semi-micro HPLC column and a linear solvent gradient. A total ion chromatogram and MRM profiles of CDU and its metabolites using the described ESI LC/MS-MS conditions are shown in Fig. 2. Although baseline resolution of CUDA, CHDU, and CODU was not achieved, the specific detection of each analyte was accomplished using MRM scans. Good linearities were observed for CDU and CUDA between the concentration and the response corresponding to the peak area ratios in the concentration range from 2 to 400 ng/ml (r ≥ 0.999). The concentration of CODU and CHDU was estimated using the relative response factor of CUDA.
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The extraction efficiencies for each analytical target were then evaluated. When 50 to 250 ng/ml CDU was spiked into rat hepatic microsomes, extraction recoveries were 101.9 ± 5.9 to 107.7 ± 4.6%. Extraction recovery of CUDA was 88.0 ± 5.0%. Since CHDU and CODU polarities are intermediate to that of CDU and CUDA, we assume that the extraction efficiencies for these primary metabolites are at least equal to that of CUDA.
Hepatic Microsomal Metabolism of CDU. The time-dependent formation
of CDU metabolites in rat and human hepatic microsomes are shown in
Fig. 3. When 1 µM CDU was
incubated in rat microsomes at 37°C for 1 h, 70% of the CDU was
metabolized within 10 min of incubation. On the other hand, over 90% of the
CUDA in a 1 µM solution remained after 1 h of incubation with rat
microsomes (data not shown). After 10 min of incubation, 312 ng/ml CDU was
transformed into 90, 163, 36, and 17 ng/ml CDU, CHDU, CODU, and CUDA
(accounting for 
95% of the initial mass), respectively. The concentration
of CHDU decreased slightly after 10 min, and the concentrations of CODU and
CUDA gradually increased until 60 min. To measure the in vitro metabolic rate,
the kinetics of the NADPH-dependent metabolism of 100 to 3000 nM CDU to CHDU
was examined with rat hepatic microsomes. The calculated kinetics parameters
of Km app and Vmax app were 223 nM and
1667 pmol/min/mg protein, respectively, by Hanes-Woolf plot and
Michaelis-Menten plot. The intrinsic clearance, calculated as
Vmax/Km, was 7.47 ml/min/mg for CHDU.
Although the relative rate of CHDU formation in human and rat microsomes was
similar (Fig. 3), the CODU and
CUDA formation was significantly lower in the human tissue preparation.
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Microsomal Metabolism Increased sEH Inhibitor Potency. Incubating
CDU with microsomes and NADPH for 10 min increased the sEH inhibition potency
of the reaction mixture by 10% (Fig.
4A). Similarly, the sEH inhibition potency of solutions containing
between 10 and 100 nM CDU were increased when incubated for 30 min with
microsomes (Fig. 4B). On the
other hand, sEH inhibition by CUDA was not influenced by either addition of
microsomes or incubation time.
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Metabolic Pathway Elucidation. The identified CDU metabolites suggested that the initial reaction leading to CHDU synthesis was a process that was most likely dependent on an enzyme or enzymes in the P450 4A family. To further implicate the P450 dependence, 1 µM CDU solution was incubated with rat microsomes for 10 min, purged with carbon monoxide for 1 min, and incubated for an additional 50 min at 37°C. The relative responses of CDU and CHDU against the internal standard (CTU) peak area were measured by LC/MS-MS. As shown in Fig. 5, the loss of CDU and the production of both CHDU and CUDA from CDU were significantly decreased upon microsome exposure to carbon monooxide. As further evidence of a P450 4A-mediated process, the CDU metabolite formation in noninduced and clofibrate-induced rat microsomes was compared (Fig. 6A). Not only was CDU metabolized to a greater extent in the clofibrate-induced microsomes, but the formation of CHDU, CODU, and CUDA was significantly (P < 0.01) higher, about 1.9-, 3.0-, and 2.8-fold, than that in noninduced microsomes. The effect of ABT on CDU metabolism was investigated (Fig. 6B). The microsomes were preincubated with 10 mM ABT for 10 min and further incubated for 20 min. The percentages of residual CDU increased with added ABT, whereas the percentages of CUDA formation significantly (P < 0.05) decreased with added ABT.
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The transformation of the -hydroxylate to the terminal aldehyde
moiety could be accomplished through either an NADPH-dependent P450-mediated
mechanism, an NAD-dependent alcohol dehydrogenase-mediated mechanism, or
perhaps both. To segregate these possibilities, rat microsomes were first
preincubated with 1 µM CDU in the presence of a NADPH generating system for
40 min to produce a metabolite mixture. To evaluate the P450-related alcohol
oxygenase component, this mixture was then enriched with additional rat
microsomes and a NADPH generating system, and incubated for 40 min. As shown
in Fig. 7, coincubation with
the NADPH generating system reduced CHDU concentrations and increased CUDA
concentrations, whereas the trace levels of CDU remaining were unchanged. To
evaluate the alcohol and aldehyde dehydrogenase-mediated components of CDU
metabolism, a metabolite mixture was prepared as above and enriched with fresh
rat microsomes and 2 mM NAD, and incubated for 40 min
(Fig. 8). The addition of NAD
significantly enhanced the transformation of CHDU to CUDA, doubling the
responses of both CODU and CUDA.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Di-substituted ureas have been identified as potent
inhibitors of this enzyme, with some in vivo efficacy
(Morisseau et al., 2002).
Although little is known about the pharmacokinetics of these compounds, by
understanding the dominant routes of metabolism, novel pharmacological agents
can be designed to suppress these transformations and thereby optimize in vivo
potency. To identify these routes of metabolism, we have investigated the
transformation of a model sEH inhibitor
(Morisseau et al., 2002), CDU,
in rat and human hepatic microsomes. Incubation of rat hepatic microsomes with CDU results in the formation of three major metabolites (Fig. 1). Since these compounds are nonvolatile and do not contain useful chromophores or fluorophores, analytical options for their detection and quantification are limited. However, using a highly specific and sensitive HPLC-tandem mass spectrometry system, we have developed a method for the simultaneous quantification of CDU and these metabolites. Analytical standards for the hydroxyl (CHDU) or oxo (CODU) compound metabolites could not be acquired for this study. However, upon collision-induced dissociation, both CUDA and these two metabolites produce primary fragments resulting from the loss of the aliphatic amine from the urea structure (Fig. 1). Therefore, to estimate the concentrations of these metabolites, the CUDA calibration curve was used as a pseudocalibration for CHDU and CODU. When this procedure was used to quantify residues in the time-dependent microsomal incubations experiments, the sum of the measured analytes at each time point corresponded to the initial CDU amount (Fig. 3). These results suggest that the use of CUDA calibration curves for estimating CHDU and CODU concentrations was valid and support the contention that the extraction recoveries of CHDU and CODU are similar to that of CUDA. A 30-mm reversed phase column was used to shorten analytical time, such that adequate separation of all analytes was achieved within 7 min using MRM mode detection. Using this method, we investigated the route and rate of CDU metabolism in rat hepatic microsomes.
Previously, we have reported that the metabolism of CDU in rat hepatic
microsomes was rapid (Watanabe et al., 2001). To estimate the in vitro
metabolic rate, we measured the kinetics of the NADPH-dependent metabolism of
CDU to CHDU with rat hepatic microsomes. The calculated Km
(223 nM) and Vmax (1.7 nmol/min/mg) for CDU suggest that
this compound is an excellent substrate for cytochromes P450, most likely a
CYP4A. Comparing the results to those reported by Amet et al.
(1995) for 12-laurate
hydoxylase activity (Km = 13 µM;
Vmax = 2.1 nmol/min/mg) suggests that CDU is at least a
40-fold better substrate for the rat -hydroxylase than lauric acid is
for the human -hydroxylase.
When cytochrome P450 was inhibited by purging with carbon monooxide, the
formation of the -hydroxylate of CDU was decreased (P <
0.05). Whereas carbon monoxide reduced CHDU formation by 20 to 30%, CUDA
formation was reduced 45% (Fig.
5). This result suggests that carbon monooxide may inhibit the
microsomal oxygenase or hydrogenase. The long alkyl chain (carbon = 12) of CDU
may be metabolized by the CYP4A family (Nebert et al., 1985;
Hardwick et al., 1987;
Sharma et al., 1989; Okita et
al., 2001). Indeed, lauric acid (dodecanoic acid) was the most efficiently
hydroxylated (Adas et al.,
1999), and the rank of catalytic activity was lauric > myristic
> palmitic. We also found that the sEH inhibitor with a 10-carbon alkyl
chain was also easily metabolized in rat hepatic microsomes (Watanabe et al.,
2001). Although the - and -1-hydroxylic derivatives of CDU
cannot be separated by our LC/MS-MS method
(Fig. 2), we can clarify that
the -hydroxylic derivative is the dominant hydroxylated metabolite from
CDU in rat hepatic microsomes by use of a long reversed phase column (data not
shown). As shown in Fig. 6A,
the formation of CHDU in clofibrate-induced rat microsomes was 1.9-fold higher
than that in noninduced microsomes. Moreover, we confirmed that the P450 4A1
activity, i.e., 12-laurate hydroxylase activity, of clofibrate-induced
microsomes was 7-fold higher than that of noninduced microsomes. Together,
these results suggest that CDU was metabolized to CHDU by a cytochrome P450 in
the 4A family. Moreover, the inhibition with ABT also supports our finding. As
shown in Fig. 3, CDU was
quickly metabolized to the -hydroxylate in rat hepatic microsomes, and
was further metabolized to aldehydic (CODU) and carboxylic (CUDA)
derivatives.
In human microsomes, the time-dependent formation profiles of CDU metabolites were similar to that of rat microsomes. However, only 30 to 40% formation of CODU and CUDA was observed, and the CDU profile was different from that of rat microsomes. These observations suggest that rat pharmacokinetic results may be weak predictors for human pharmacokinetics with respect to these aliphatic-containing compounds.
To confirm sEH inhibition by CDU after incubation with microsomes, the time
course of sEH inhibition by CDU and CUDA was investigated
(Fig. 4). Although 70% of
CDU was metabolized to CUDA and CHDU within 10 min of incubation
(Fig. 3), sEH inhibition was
increased by 10% (Fig. 4). In
addition, CUDA was extremely stable in rat hepatic microsomes, with over 90%
of CUDA still remaining after 60 min of incubation (data not shown). This
result suggests that not only CUDA but also the -hydroxylic derivative
(CHDU) is an excellent sEH inhibitor. These findings agree with the fact that
sEH, the pharmacological target, can accept polar functionalities that are at
a significant distance from the pharmacophore
(Morisseau et al., 2002).
The described results have been summarized into a proposed metabolic
pathway of CDU in rat hepatic microsomes
(Fig. 9). We designed two
experiments to clarify our hypothesis of a metabolic pathway from hydroxylic
derivative to carboxylic derivative (Figs.
7 and
8). The formation of aldehydic
and carboxylic derivative was gradually increased with an increase in
incubation time. On the other hand, the formation of hydroxy derivative was
rapid, and the maximum concentration was reached at 20 min of incubation. As
shown in Fig. 9, several
enzymes are associated to act in this metabolic pathway, and CUDA may be
generated by a two-step mechanism. When additional microsomes with NADPH
generating system were added to rat microsomes to enhance microsomal
cytochrome P450 activity, the hydroxy derivative was significantly decreased
without additional depletion of CDU (P < 0.05)
(Fig. 7). This finding suggests
that metabolism from hydroxylic derivative to aldehydic derivative is partly
catalyzed by microsomal cytochrome P450-related alcohol oxygenase.
Furthermore, we added NAD as a dehydrogenase cofactor
(Fig. 8). Consequently, 90% of
the hydroxy derivative was metabolized to aldehydic and carboxylic
derivatives, a significant enhancement (P < 0.01). From these
results, we can indicate that the main metabolic pathway from hydroxylic to
carboxylic derivative of CDU is catalyzed by alcohol dehydrogenase and
aldehyde dehydrogenase. Indeed, the metabolic pathway of sameridine, which is
an amide-type local anesthetic-analgesic agent with a hexyl side chain, is
similar to this pathway
(Sohlenius-Sternbeck et al.,
2000). Also, Sinz et al.
(1997) have reported that the
alkyl side chain of a fatty acid anilide is metabolized via a similar
pathway.
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In conclusion, the results suggest that CDU, which has a long alkyl chain,
is metabolized to CUDA by a multistep pathway that includes cytochrome P450
hydroxylases, alcohol dehydrogenase and microsomal cytochrome P450-related
alcohol oxygenase, and aldehyde dehydrogenase
(Tottmar et al., 1973; Koivula
et al., 1975) (Fig. 9). We
found that the activity of sEH was increased with incubation in rat hepatic
microsomes, suggesting that at least some of the metabolites of CDU are
themselves potent sEH inhibitors. The solubility of these metabolites was much
better than that of CDU. These results suggest that the synthesis of ester
derivatives of CUDA will likely yield potent sEH inhibitors with improved
aqueous solubility. In addition, the presence at the -position of
functions not transformed by P450 will decrease metabolism of sEH inhibition
and increase their in vivo stability.
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Footnotes |
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1 Abbreviations used are: EH, epoxide hydrolase(s); sEH, soluble epoxide
hydrolase; CDU, 1-cyclohexyl-3-dodecyl-urea; LC/MS-MS, high-performance liquid
chromatography with tandem mass spectrometry; CUDA,
(12-(3-cyclohexyl-ureido)-dodecanoic acid; CTU,
1-cyclohexyl-3–tetradecyl-urea; CHDU,
1-cyclohexyl-3-(12-hydroxy-dodecyl)-urea; CODU,
1-cyclohexyl-3-(12-oxo-dodecyl)-urea; P450, cytochrome P450; ABT,
aminobenzotriazol; ESI, electrospray ionization; HPLC, high-performance liquid
chromatography; MRM, multiple reaction monitoring. 
Address correspondence to: Bruce D. Hammock, Department of Entomology & Cancer Research Center, University of California, Davis, CA 95616. E-mail: bdhammock{at}ucdavis.edu
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-1)-hydroxylation of lauric acid
as an in vitro substrate probe for human liver CYP2E1. Biochem
Pharmacol 50:
1775–1782.[CrossRef][Medline]
-hydroxylase (cytochrome P-450LAW).
J Biol Chem 262:
801–810.This article has been cited by other articles:
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  J. D. Imig, X. Zhao, C. Z. Zaharis, J. J. Olearczyk, D. M. Pollock, J. W. Newman, I.-H. Kim, T. Watanabe, and B. D. Hammock An Orally Active Epoxide Hydrolase Inhibitor Lowers Blood Pressure and Provides Renal Protection in Salt-Sensitive Hypertension Hypertension, October 1, 2005; 46(4): 975 - 981. [Abstract] [Full Text] [PDF]
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 X. Zhao, T. Yamamoto, J. W. Newman, I.-H. Kim, T. Watanabe, B. D. Hammock, J. Stewart, J. S. Pollock, D. M. Pollock, and J. D. Imig Soluble Epoxide Hydrolase Inhibition Protects the Kidney from Hypertension-Induced Damage J. Am. Soc. Nephrol., May 1, 2004; 15(5): 1244 - 1253. [Abstract] [Full Text] [PDF]
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, P <
0.05 significantly different from control.
