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Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)- cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke. Am J Physiol Heart Circ Physiol 305: H1605–H1613, 2013. First published September... more
Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-
cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic
stroke. Am J Physiol Heart Circ Physiol 305: H1605–H1613, 2013. First
published September 16, 2013; doi:10.1152/ajpheart.00471.2013.—
Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to
inactive dihydroxy metabolites. The primary goals of this study were to
investigate the effects of acute sEH inhibition by trans-4-[4-(3-
adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow
(CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was
induced in rats for 90 min followed by reperfusion. At the end of 24 h
after reperfusion rats were euthanized for infarct volume assessment by
triphenyltetrazolium chloride staining. Brain cortical sEH activity was
assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was
evaluated by arm flexion and sticky-tape tests. Changes in CBF were
assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at
180 min after reperfusion. Neuroprotective effects of t-AUCB were
evaluated in primary rat neuronal cultures by Cytotox-Flour kit and
propidium iodide staining. t-AUCB significantly reduced cortical infarct
volume by 35% (14.5 2.7% vs. 41.5 4.5%), elevated cumulative
epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain
cortex by twofold (4.40 1.89 vs. 1.97 0.85), and improved functional
outcome in arm-flexion test (day 1: 3.28 0.5 s vs. 7.50 0.9 s; day 2:
1.71 0.4 s vs. 5.28 0.5 s) when compared with that of the
vehicle-treated group. t-AUCB significantly reduced neuronal cell death
in a dose-dependent manner (vehicle: 70.9 7.1% vs. t-AUCB0.1M:
58 5.11% vs. t-AUCB0.5M: 39.9 5.8%). These findings suggest that
t-AUCB may exert its neuroprotective effects by affecting multiple
components of neurovascular unit including neurons, astrocytes, and
microvascular flow
cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic
stroke. Am J Physiol Heart Circ Physiol 305: H1605–H1613, 2013. First
published September 16, 2013; doi:10.1152/ajpheart.00471.2013.—
Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to
inactive dihydroxy metabolites. The primary goals of this study were to
investigate the effects of acute sEH inhibition by trans-4-[4-(3-
adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow
(CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was
induced in rats for 90 min followed by reperfusion. At the end of 24 h
after reperfusion rats were euthanized for infarct volume assessment by
triphenyltetrazolium chloride staining. Brain cortical sEH activity was
assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was
evaluated by arm flexion and sticky-tape tests. Changes in CBF were
assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at
180 min after reperfusion. Neuroprotective effects of t-AUCB were
evaluated in primary rat neuronal cultures by Cytotox-Flour kit and
propidium iodide staining. t-AUCB significantly reduced cortical infarct
volume by 35% (14.5 2.7% vs. 41.5 4.5%), elevated cumulative
epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain
cortex by twofold (4.40 1.89 vs. 1.97 0.85), and improved functional
outcome in arm-flexion test (day 1: 3.28 0.5 s vs. 7.50 0.9 s; day 2:
1.71 0.4 s vs. 5.28 0.5 s) when compared with that of the
vehicle-treated group. t-AUCB significantly reduced neuronal cell death
in a dose-dependent manner (vehicle: 70.9 7.1% vs. t-AUCB0.1M:
58 5.11% vs. t-AUCB0.5M: 39.9 5.8%). These findings suggest that
t-AUCB may exert its neuroprotective effects by affecting multiple
components of neurovascular unit including neurons, astrocytes, and
microvascular flow
Research Interests:
A specific, accurate, precise and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of five 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase... more
A specific, accurate, precise and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of five 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase
inhibitors, viz. atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin, in pharmaceutical formulations and extended the
application to in vitro metabolism studies of these statins. Ternary gradient elution at a flow rate of 1 mL/min was employed on
an Intertisl ODS 3V column (4.6 × 250 mm, 5 µm) at ambient temperature. The mobile phase consisted of 0.01 M ammonium acetate (pH 5.0), acetonitrile and methanol. Theophylline was used as an internal standard (IS). The HMG-CoA reductase inhibitors
and their metabolites were monitored at a wavelength of 237 nm. Drugs were found to be 89.6–105.6% of their label’s claim in
the pharmaceutical formulations. For in vitro metabolism studies the reaction mixtures were extracted with simple liquid–liquid
extraction using ethyl acetate. Baseline separation of statins and their metabolites along with IS free from endogenous interferences was achieved. Nominal retention times of IS, atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were 7.5, 17.2,
21.6, 28.5, 33.5 and 35.5 min, respectively. The proposed method is simple, selective and could be applicable for routine analysis of
HMG-CoA reductase inhibitors in pharmaceutical preparations as well as in vitro metabolism studies
inhibitors, viz. atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin, in pharmaceutical formulations and extended the
application to in vitro metabolism studies of these statins. Ternary gradient elution at a flow rate of 1 mL/min was employed on
an Intertisl ODS 3V column (4.6 × 250 mm, 5 µm) at ambient temperature. The mobile phase consisted of 0.01 M ammonium acetate (pH 5.0), acetonitrile and methanol. Theophylline was used as an internal standard (IS). The HMG-CoA reductase inhibitors
and their metabolites were monitored at a wavelength of 237 nm. Drugs were found to be 89.6–105.6% of their label’s claim in
the pharmaceutical formulations. For in vitro metabolism studies the reaction mixtures were extracted with simple liquid–liquid
extraction using ethyl acetate. Baseline separation of statins and their metabolites along with IS free from endogenous interferences was achieved. Nominal retention times of IS, atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were 7.5, 17.2,
21.6, 28.5, 33.5 and 35.5 min, respectively. The proposed method is simple, selective and could be applicable for routine analysis of
HMG-CoA reductase inhibitors in pharmaceutical preparations as well as in vitro metabolism studies
Research Interests:
The metabolites of arachidonic acid (AA) produced from the cyclooxygenase (COX) pathway, collectively termed as prostanoids, and from the CYP 450 pathway, eicosanoids, have been implicated in various neuro-degenerative and... more
The metabolites of arachidonic acid (AA) produced from the cyclooxygenase (COX) pathway, collectively termed as prostanoids, and from the CYP 450 pathway, eicosanoids, have been implicated
in various neuro-degenerative and neuroinflammatory diseases. This study developed a quantitative
UPLC–MS/MS method to simultaneously measure 11 prostanoids including prostaglandins and cyclopentenone metabolites in the rat brain cortical tissue. Linear calibration curves ranging from 0.104 to
33.3 ng/ml were validated. The inter-day and intra-day variance for all metabolites was less than 15%.
The extraction recovery efficiency and matrix (deionized water) effects measured at 12.5 ng/ml (750 pg
on column) ranged from 88 to 100% and 3 to 14%, respectively, with CV% values below 20%. Additionally,
applying the processing and extraction conditions of this method to our previous CYP450 eicosanoids
method resulted in overall improvement in extraction recovery and reduction in matrix effects at low
(0.417 ng/ml) and high (8.33 ng/ml) concentrations. In rat brain cortical tissue samples, concentrations
of prostanoids ranged from 10.2 to 937 pmol/g wet tissue and concentration of eicosanoids ranged from
2.23 to 793 pmol/g wet tissue. These data demonstrate that the successive measurement of prostanoids
and eicosanoids from a single extracted sample of rat brain tissue can be achieved with a UPLC–MS/MS
system and that this method is necessary for evaluation of these metabolites to delineate their role in
various neuroinflammatory and cerebrovascular disorders
in various neuro-degenerative and neuroinflammatory diseases. This study developed a quantitative
UPLC–MS/MS method to simultaneously measure 11 prostanoids including prostaglandins and cyclopentenone metabolites in the rat brain cortical tissue. Linear calibration curves ranging from 0.104 to
33.3 ng/ml were validated. The inter-day and intra-day variance for all metabolites was less than 15%.
The extraction recovery efficiency and matrix (deionized water) effects measured at 12.5 ng/ml (750 pg
on column) ranged from 88 to 100% and 3 to 14%, respectively, with CV% values below 20%. Additionally,
applying the processing and extraction conditions of this method to our previous CYP450 eicosanoids
method resulted in overall improvement in extraction recovery and reduction in matrix effects at low
(0.417 ng/ml) and high (8.33 ng/ml) concentrations. In rat brain cortical tissue samples, concentrations
of prostanoids ranged from 10.2 to 937 pmol/g wet tissue and concentration of eicosanoids ranged from
2.23 to 793 pmol/g wet tissue. These data demonstrate that the successive measurement of prostanoids
and eicosanoids from a single extracted sample of rat brain tissue can be achieved with a UPLC–MS/MS
system and that this method is necessary for evaluation of these metabolites to delineate their role in
various neuroinflammatory and cerebrovascular disorders
Research Interests:
A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle... more
A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and
COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural
changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly.
Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo.
Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was
identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model
COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural
changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly.
Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo.
Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was
identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model
Research Interests:
Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with... more
Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite
i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved
deproteinization of 500 L of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 L acetonitrile
containing 25% perchloric acid. An aliquot of 100 L supernatant was injected onto a C18 column. The chromatographic separation was achieved
by gradient elution consisting of 0.05 M formic acid:acetonitrile:methanol:water at a flow rate of 1.0 mL/min. The detection of analyte peaks were
achieved by monitoring the eluate using an UV detector set at 250 nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide
(EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56 min, respectively. Average extraction efficiencies of EZM, EZM-G and IS
was >75–80% and for EZM-K was >50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02 g/mL.
Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the
method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies
i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved
deproteinization of 500 L of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 L acetonitrile
containing 25% perchloric acid. An aliquot of 100 L supernatant was injected onto a C18 column. The chromatographic separation was achieved
by gradient elution consisting of 0.05 M formic acid:acetonitrile:methanol:water at a flow rate of 1.0 mL/min. The detection of analyte peaks were
achieved by monitoring the eluate using an UV detector set at 250 nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide
(EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56 min, respectively. Average extraction efficiencies of EZM, EZM-G and IS
was >75–80% and for EZM-K was >50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02 g/mL.
Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the
method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies
Research Interests:
Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived... more
Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived
20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the
role of CYP4F2 in vascular disease, we generated mice
with endothelial expression of human CYP4F2 (Tie2-
CYP4F2-Tr). LC/MS/MS analysis revealed 2-fold
increases in 20-HETE levels in tissues and endothelial
cells (ECs), relative to wild-type (WT) controls. Tie2-
CYP4F2-Tr ECs demonstrated increases in growth
(267.133.4 vs. 205.013% at 48 h) and tube formation (7.71.1 vs. 1.60.5 tubes/field) that were 20-
HETE dependent and associated with up-regulation of
prooxidant NADPH oxidase and proangiogenic VEGF.
Increases in VEGF and NADPH oxidase levels were
abrogated by inhibitors of NADPH oxidase and MAPK,
respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-
CYP4F2-Tr mice (18.62.7 vs. 7.92.7 pg/ml) were
up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and
blood pressure were unchanged. Our findings indicate
that human CYP4F2 significantly increases 20-HETE
production, CYP4F2-derived 20-HETE mediates EC
proliferation and angiogenesis via VEGF- and NADPH
oxidase-dependent manners, and the Tie2-CYP4F2-Tr
mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the
vasculature.—Cheng, J., Edin, M. L., Hoopes, S. L., Li,
H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih,
F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake,
G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of
mice with endothelial expression of cytochrome P450
4F2
20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the
role of CYP4F2 in vascular disease, we generated mice
with endothelial expression of human CYP4F2 (Tie2-
CYP4F2-Tr). LC/MS/MS analysis revealed 2-fold
increases in 20-HETE levels in tissues and endothelial
cells (ECs), relative to wild-type (WT) controls. Tie2-
CYP4F2-Tr ECs demonstrated increases in growth
(267.133.4 vs. 205.013% at 48 h) and tube formation (7.71.1 vs. 1.60.5 tubes/field) that were 20-
HETE dependent and associated with up-regulation of
prooxidant NADPH oxidase and proangiogenic VEGF.
Increases in VEGF and NADPH oxidase levels were
abrogated by inhibitors of NADPH oxidase and MAPK,
respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-
CYP4F2-Tr mice (18.62.7 vs. 7.92.7 pg/ml) were
up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and
blood pressure were unchanged. Our findings indicate
that human CYP4F2 significantly increases 20-HETE
production, CYP4F2-derived 20-HETE mediates EC
proliferation and angiogenesis via VEGF- and NADPH
oxidase-dependent manners, and the Tie2-CYP4F2-Tr
mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the
vasculature.—Cheng, J., Edin, M. L., Hoopes, S. L., Li,
H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih,
F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake,
G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of
mice with endothelial expression of cytochrome P450
4F2
Research Interests:
Tuberculosis continues to be a major cause of morbidity and mortality all over the world. Various 7-substituted ciprofloxacin derivatives were synthesized and evaluated for antimycobacterial activity in vitro and in vivo against... more
Tuberculosis continues to be a major cause of morbidity and mortality all over the world. Various 7-substituted ciprofloxacin derivatives were synthesized and evaluated for antimycobacterial activity in vitro and in vivo against Mycobacterium tuberculosis and for inhibition of the supercoiling activity of DNA gyrase from Mycobacterium smegmatis. Preliminary results indicated
that most of the compounds demonstrated better in vitro antimycobacterial activity against M. tuberculosis than ciprofloxacin.
Compound 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-[10-(5-methylisatinyl-b-semicarbazo)]methyl]N1-piperazinyl]-3-quinoline carboxylic acid (3h) decreased the bacterial load in spleen tissue with 0.76-log10 protections and was considered to be moderately
active in reducing bacterial count in spleen. The results demonstrated the potential and importance of developing new quinolone
derivatives against mycobacterial infections
that most of the compounds demonstrated better in vitro antimycobacterial activity against M. tuberculosis than ciprofloxacin.
Compound 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-[[N4-[10-(5-methylisatinyl-b-semicarbazo)]methyl]N1-piperazinyl]-3-quinoline carboxylic acid (3h) decreased the bacterial load in spleen tissue with 0.76-log10 protections and was considered to be moderately
active in reducing bacterial count in spleen. The results demonstrated the potential and importance of developing new quinolone
derivatives against mycobacterial infections
Research Interests:
Ciclesonide, a novel glucocorticosteroid, through a rapid metabolism to desisobutyryl-ciclesonide (des-ciclesonide), provides an effective treatment option for asthma episodes by the inhaled route of administration. The availability of... more
Ciclesonide, a novel glucocorticosteroid, through a rapid metabolism to desisobutyryl-ciclesonide
(des-ciclesonide), provides an effective treatment option for asthma episodes by the inhaled route of
administration. The availability of pharmacokinetic parameters (clearance [CL/F]; volume of
distribution [Vd/F]; elimination half-life [T1/2]; and elimination rate constant [Kel]) in mice, rats,
rabbits, and dogs enabled the prediction of human parameter values for des-ciclesonide using the
well-accepted tool of allometry after intravenous administration of ciclesonide. However, as a result
of the rapid conversion of ciclesonide, it was possible to perform allometry for the CL parameter
only. Simple allometry (CL = 4.781W0.7874; R2 = 0.9968) appeared to predict the CL of ciclesonide in
close proximity of the observed value (observed: 101.25 L/h versus predicted: 135.62 L/h). In
a similar manner, simple allometry predicted the human pharmacokinetic parameters of desciclesonide (CL/F, Vd/F, T1/2, and Kel) within a two- to threefold range of the observed values. The
allometric equations for des-ciclesonide parameter values were: CL/F = 4.8166W0.492 (R2 = 0.8598);
Vd/F = 19.052W0.632 (R2 = 0.9049); T1/2 = 3.7598W20.1611(R2 = 0.8551); and Kel = 0.1832W0.1596 (R2 =
0.8632). In conclusion, the data suggested that allometry tool may be amenable for the prediction of
the pharmacokinetic parameters of des-ciclesonide despite differences in the conversion rates and
bioavailability of the active metabolite in various animal species
(des-ciclesonide), provides an effective treatment option for asthma episodes by the inhaled route of
administration. The availability of pharmacokinetic parameters (clearance [CL/F]; volume of
distribution [Vd/F]; elimination half-life [T1/2]; and elimination rate constant [Kel]) in mice, rats,
rabbits, and dogs enabled the prediction of human parameter values for des-ciclesonide using the
well-accepted tool of allometry after intravenous administration of ciclesonide. However, as a result
of the rapid conversion of ciclesonide, it was possible to perform allometry for the CL parameter
only. Simple allometry (CL = 4.781W0.7874; R2 = 0.9968) appeared to predict the CL of ciclesonide in
close proximity of the observed value (observed: 101.25 L/h versus predicted: 135.62 L/h). In
a similar manner, simple allometry predicted the human pharmacokinetic parameters of desciclesonide (CL/F, Vd/F, T1/2, and Kel) within a two- to threefold range of the observed values. The
allometric equations for des-ciclesonide parameter values were: CL/F = 4.8166W0.492 (R2 = 0.8598);
Vd/F = 19.052W0.632 (R2 = 0.9049); T1/2 = 3.7598W20.1611(R2 = 0.8551); and Kel = 0.1832W0.1596 (R2 =
0.8632). In conclusion, the data suggested that allometry tool may be amenable for the prediction of
the pharmacokinetic parameters of des-ciclesonide despite differences in the conversion rates and
bioavailability of the active metabolite in various animal species
Research Interests:
The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver... more
The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism,
oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX
was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without
ABT. The enzyme kinetics (Vmax and Km) of the formation of 6-hydroxychlorzoxazone (OH-CZX)
was found to be similar among rat liver microsomes (3486 pmol mg protein1 min1 and 345 mM),
human liver microsomes (3194 pmol mg protein1 min1 and 335 mM) and pure isozyme
(3423 pmol mg protein1 min1 and 403 mM), but KI and Kinact values for ABT towards the ability
to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 mM
and 0.12 min1; human: 27.19 mM and 0.14 min1) and pure isozyme (3.18 mM and 0.29 min1).
The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment
of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance
(threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats
were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV
administration; however, we were able to measure OH-CZX in rats administered with CZX by oral
and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was 71% when
dosed alone and reached 100% under pre-treatment with ABT. The t1/2 values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT,
suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating
that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations
of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h
post-treatment
oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX
was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without
ABT. The enzyme kinetics (Vmax and Km) of the formation of 6-hydroxychlorzoxazone (OH-CZX)
was found to be similar among rat liver microsomes (3486 pmol mg protein1 min1 and 345 mM),
human liver microsomes (3194 pmol mg protein1 min1 and 335 mM) and pure isozyme
(3423 pmol mg protein1 min1 and 403 mM), but KI and Kinact values for ABT towards the ability
to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 mM
and 0.12 min1; human: 27.19 mM and 0.14 min1) and pure isozyme (3.18 mM and 0.29 min1).
The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment
of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance
(threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats
were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV
administration; however, we were able to measure OH-CZX in rats administered with CZX by oral
and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was 71% when
dosed alone and reached 100% under pre-treatment with ABT. The t1/2 values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT,
suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating
that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations
of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h
post-treatment
Research Interests:
Vasoconstrictive and vasodilatory eicosanoids generated after cardiac arrest (CA) may contribute to cerebral vasomotor disturbances and neurodegeneration. We evaluated the balance of vasodilator/vasoconstrictor eicosanoids produced by... more
Vasoconstrictive and vasodilatory eicosanoids generated after cardiac arrest (CA) may contribute to cerebral vasomotor
disturbances and neurodegeneration. We evaluated the balance of vasodilator/vasoconstrictor eicosanoids produced by
cytochrome P450 (CYP) metabolism, and determined their role on cortical perfusion, functional outcome, and neurodegeneration
after pediatric asphyxial CA. Cardiac arrest of 9 and 12 minutes was induced in 16- to 18-day-old rats. At 5 and 120 minutes after CA,
we quantified the concentration of CYP eicosanoids in the cortex and subcortical areas. In separate rats, we inhibited
20-hydroxyeicosatetraenoic acid (20-HETE) synthesis after CA and assessed cortical cerebral blood flow (CBF), neurologic deficit
score, neurodegeneration, and edema. After 9 minutes of CA, vasodilator eicosanoids markedly increased versus sham. Conversely,
after 12 minutes of CA, vasoconstrictor eicosanoid 20-HETE increased versus sham, without compensatory increases in vasodilator
eicosanoids. Inhibition of 20-HETE synthesis after 12 minutes of CA decreased cortical 20-HETE levels, increased CBF, reduced
neurologic deficits at 3 hours, and reduced neurodegeneration and edema at 48 hours versus vehicle-treated rats. In conclusion,
cerebral vasoconstrictor eicosanoids increased after a pediatric CA of 12 minutes. Inhibition of 20-HETE synthesis improved cortical
perfusion and short-term neurologic outcome. These results suggest that alterations in CYP eicosanoids have a role in cerebral
hypoperfusion and neurodegeneration after CA and may represent important therapeutic targets
disturbances and neurodegeneration. We evaluated the balance of vasodilator/vasoconstrictor eicosanoids produced by
cytochrome P450 (CYP) metabolism, and determined their role on cortical perfusion, functional outcome, and neurodegeneration
after pediatric asphyxial CA. Cardiac arrest of 9 and 12 minutes was induced in 16- to 18-day-old rats. At 5 and 120 minutes after CA,
we quantified the concentration of CYP eicosanoids in the cortex and subcortical areas. In separate rats, we inhibited
20-hydroxyeicosatetraenoic acid (20-HETE) synthesis after CA and assessed cortical cerebral blood flow (CBF), neurologic deficit
score, neurodegeneration, and edema. After 9 minutes of CA, vasodilator eicosanoids markedly increased versus sham. Conversely,
after 12 minutes of CA, vasoconstrictor eicosanoid 20-HETE increased versus sham, without compensatory increases in vasodilator
eicosanoids. Inhibition of 20-HETE synthesis after 12 minutes of CA decreased cortical 20-HETE levels, increased CBF, reduced
neurologic deficits at 3 hours, and reduced neurodegeneration and edema at 48 hours versus vehicle-treated rats. In conclusion,
cerebral vasoconstrictor eicosanoids increased after a pediatric CA of 12 minutes. Inhibition of 20-HETE synthesis improved cortical
perfusion and short-term neurologic outcome. These results suggest that alterations in CYP eicosanoids have a role in cerebral
hypoperfusion and neurodegeneration after CA and may represent important therapeutic targets