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Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania (S.M.P., T.M.M., M.M.K., Y.M., L.C.R.); Magee-Womens Research Institute, Pittsburgh, Pennsylvania (L.C.R.); Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (S.H.G.); and Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania (S.H.G.)
(Received June 27, 2008; Accepted August 21, 2008)
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Abstract |
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; Kalsotra et al., 2007). In several tissues, such as the kidney and brain, the predominant P450 isoforms expressed are involved in endogenous substrate bioactivation (Meyer et al., 2007), rather than xenobiotic metabolism.
One such role for the P450 enzyme system in endogenous substrate bioactivation is the mono-oxygenation of arachidonic acid to form potent vasoactive eicosanoids. Specifically, P450 enzymes catalyze the epoxygenation at the double bonds of arachidonic acid to form epoxyeicosatrienoic acids (EETs) (Luo et al., 1998). P450 enzymes also catalyze the hydroxylation of arachidonic acid on the terminal carbons to form several hydroxyeicosatetraenoic acids (HETEs). EET and HETE metabolites produce a growing number of effects on vascular smooth muscle and other tissues. Specifically, the terminal hydroxylation of arachidonic acid to form 20-HETE produces potent microvascular vasoconstriction (Harder et al., 1994), mediates angiogenic effects (Amaral et al., 2003), and has been shown to augment vascular smooth muscle cell migration (Stec et al., 2007). Collectively, these studies suggest that the mono-oxygenation pathways of arachidonic acid metabolism are highly potent regulators of microvascular tone and growth.
Growing evidence has implicated 20-HETE in the pathogenesis of cardiovascular and neurovascular disease. Animal studies have demonstrated that inhibition of 20-HETE formation is neuroprotective in temporary focal ischemia and subarachnoid hemorrhage models (Takeuchi et al., 2005; Omura et al., 2006; Poloyac et al., 2006), thereby implicating 20-HETE as a mediator of ischemic tissue damage. Clinical studies evaluating polymorphisms in the critical enzymes that control 20-HETE production are also supportive of a role for this mono-oxygenated metabolite in diseases of cardiovascular and neurovascular origin (Gainer et al., 2005; Mayer et al., 2006). Similarly, prior studies in our laboratory have demonstrated that 20-HETE is also found in human cerebrospinal fluid after subarachnoid hemorrhage (Poloyac et al., 2005).
Because of the multitude of actions of 20-HETE, specific chemical inhibitors are in development to elucidate the role of 20-HETE in disease pathogenesis. The majority of 20-HETE inhibitors have targeted the enzymatic formation by the CYP4A and CYP4F isoforms. These 20-HETE inhibitors include 17-octadecynoic acid (17-ODYA), N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine (HET0016), and, more recently, N-(3-chloro-4-morpholin-4-yl) phenyl-N'hydroxyimidoformamide (TS-011) (Miyata et al., 2005; Omura et al., 2006). Of these inhibitors, the HET0016 and TS-011 compounds share similar structural characteristics and presumably similar mechanisms of P450 enzyme inhibition (Nakamura et al., 2003; Seki et al., 2005). HET0016 is a specific, commercially available inhibitor of 20-HETE. Because of its specificity, potency, and availability, HET0016 is being used as an experimental tool to determine the in vivo and in vitro role of 20-HETE formation in various disease states.
One of the limitations of the use of HET0016 for in vivo studies has been the poor aqueous solubility of the compound and the limited knowledge about the time course and mechanism of inhibition. Furthermore, little information exists about the tissue selectivity and the in vivo concentration necessary for inhibition of 20-HETE in the rat brain tissues. To better understand the pharmacologic utility of HET0016, our laboratory set out to elucidate the effects of HET0016 on the enzymatic formation of 20-HETE in the rat brain. A secondary purpose of this work was to determine the dose/concentration response relationship for 20-HETE inhibition in the rat brain.
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Materials and Methods |
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Animals. Male Sprague-Dawley rats (b.wt., 250-300 g) were obtained from Hilltop Laboratory Animals Inc. (Scottsdale, PA). The animals were maintained on a 12-h light/dark cycle and were given free access to pellets and water. Rats were placed under light anesthesia with 3:1 ketamine and xylazine (v/v) (Webster Veterinary Supply, Sterling, MA) or with the use of a spontaneous inhalational anesthetic system [SurgiVet (Smiths Medical, Waukesha, WI) V7216 Isotec 4] using isoflurane in conjunction with pure oxygen and nitrous oxide. Animals were sacrificed by decapitation, and brain cortical tissue was excised. A separate set of animals received bilateral temporary femoral cannulas via insertion of 50-gauge polyethylene tubing. A single intravenous (i.v.) injection of HET0016 1 mg/kg in 15% HPβCD or vehicle was administered via one of the two femoral cannulas, and blood samples were obtained via the opposite femoral cannula for serum isolation. Animals were sacrificed at multiple time points and brain cortical tissue was harvested. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.
Solubility. To determine the solubility of HET0016, 0.5 mg was added to 1 ml of distilled, deionized water (ddH2O) and 0.5 mg was added to 1 ml of 15% HPβCD (n = 6). Both groups agitated for 48 h at room temperature on a Titer Plate Shaker (Lab-Line Instruments, Inc., Melrose Park, IL) set at a speed of 450 rpm. At the end of the agitation period, both groups were lyophilized to complete dryness using a FreeZone 6 Lyopholizer (Labconco, Inc., Kansas City, MO), reconstituted in 1 ml of ddH2O, and syringe-filtered using Millipore 0.45 µm, 13 mm polyvinylidene difluoride Syringe Filters (Thermo Fisher Scientific). HET0016 HPβCD solubility was determined by adding varying amounts of HET0016 to 1 ml of 15% HPβCD. The amounts of HET0016 used were 0.5, 0.75, 1, 1.25, 1.5, 1.75, and 2 mg (n = 3). After the addition of HET0016, samples were isolated and lyophilized for subsequent HET0016 concentration determination.
Formulation. Formulation of HET0016 with HPβCD was achieved by adding 0.5 mg of HET0016 to 1 ml of 15% HPβCD and agitating at room temperature for 48 h on a Titer Plate Shaker (Lab-Line Instruments, Inc.) set at a speed of 450 rpm. At the end of the agitation period, the samples were lyophilized to complete dryness with a FreeZone 6 Lyopholizer (Labconco, Inc.). Samples were reconstituted with phosphate-buffered saline and syringe filtered using Millipore 0.45 µm, 13 mm polyvinylidene difluoride Syringe Filters (Thermo Fisher Scientific) before administering to animals.
Microsomal Preparation. Brain cortical tissue was homogenized in cold buffer (50 mM tris buffer, 150 mM KCl, 0.1 mM dithiothreitol, 1 mM EDTA, and 20% glycerol, pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride and 0.113 mM butylated hydroxytoluene. Microsomes were obtained via differential centrifugation in a Beckman Coulter Optima XL-100K ultracentrifuge (Beckman Coulter, Fullerton, CA) as previously described by Rockich and Blouin (1999). Total microsomal protein was determined by the method detailed by Lowry et al. (1951).
Cortical Microsome Inhibition Study. Brain cortical microsomes from untreated animals were incubated with HET0016 (48.5 nM) with and without a 10 min preincubation step to determine the mechanism of inhibition. Incubations, containing 320 µg of microsomal protein, consisted of 100 µM of arachidonic acid in a 1 ml volume of incubation buffer (0.12 M potassium phosphate buffer containing 5 mM magnesium chloride). Reactions were initiated by the addition of 1 mM NADPH with a subsequent 1 mM concentration added 30 min afterwards. Incubations were carried out at 37°C for 60 min and the reaction was stopped by placing the tubes on ice. To each sample, 75 ng of 20-HETE-d6 was added as the internal standard (IS). Microsomal incubations were extracted twice with three milliliters of diethyl ether, dried under nitrogen gas, and reconstituted in 80:20 methanol/deionized water.
In a subsequent study, brain cortical microsomes from untreated animals were incubated with or without HET0016 (24.2 nM) and analyzed for 20-HETE formation under varying substrate concentrations. Incubations, containing 325 µg of microsomal protein, were conducted over arachidonic acid concentrations ranging from 0.2 to 120 µM in a 1-ml volume of incubation buffer, and the reaction was carried out as previously described.
Incubations of brain cortical microsomes prepared from vehicle-treated or HET0016-treated animals (n = 6 per treatment group) were carried out using 325 µg of microsomal protein. Tissue was harvested 1 h after i.v. dose administration for subsequent ex vivo assessment of 20-HETE formation. Brain microsomes were incubated in the presence of 50 µM arachidonic acid to determine the degree of 20-HETE formation.
Tissue Extraction. 20-HETE and HET0016 concentrations were determined in brain cortical tissue samples of vehicle-treated and inhibitor-treated animals using solid phase extraction as previously described by Poloyac et al. (2004) with minor modifications. Briefly, tissue samples were homogenized in a 0.12 M potassium phosphate buffer containing 5 mM magnesium chloride and 0.113 mM butylated hydroxytoluene and centrifuged for 30 min at 10,000 rpm. The supernatant was removed, 7.5 ng of 20-HETE-d6 and 6.6 ng of N1-(4-butyl-2-methylphenyl) acetamide (Maybridge, Cambridge, UK) were added as the internal standards, and samples were extracted using hydrophilic lipophilic balanced solid phase extraction cartridges (Oasis; Waters, Milford, MA). Columns were washed with three 1-ml volumes of 5% methanol and were eluted with 100% methanol. Extracts were dried under nitrogen gas at 37°C and reconstituted in 200 µl of 80:20 methanol/deionized water.
HET0016 Validation. Validation of the quantitative assay was performed by using eight standard concentrations of HET0016, prepared in a 1-ml volume of incubation buffer, and extracted via solid phase extraction using the above method. The amounts of HET0016 in the standards ranged from 0.05 to 2 ng injected on column. Duplicate standard curves were prepared and analyzed over three runs. Curves were calculated based on the peak-area ratios of HET0016 to the internal standard and plotted against the amount of HET0016 injected onto the column. Calibration curves were generated by weighted (1/Y) linear regression. Precision and accuracy were determined by the analysis of HET0016 quality control (QC) samples. HET0016 was spiked into buffer to yield low, medium, and high QCs, corresponding to 0.09, 0.45, and 1.25 ng on column, respectively. Six samples at each level were analyzed for 2 days, followed by 12 replicates of each on the final day of validation. Accuracy was determined by comparing the concentrations measured in the quality control samples with respect to known values and expressed as relative S.E. (% bias). Precision was evaluated by measuring separately prepared QCs and expressed as relative S.D. of the mean concentrations. Recovery of HET0016 was estimated by comparing the area ratios of extracted samples to those of neat samples prepared in respective concentrations in 80:20 methanol/deionized water.
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Separation of HET0016 was conducted using the above methods with a slight modification in the gradient. From an initial mixture of 40:60 A/B, mobile phase B increased from 60 to 95% in a linear gradient over 11 min, ramped again up to 98% over 1 min, and remained there over the next 4 min. This was followed by a linear return to initial conditions over 0.2 min with a 3-min pre-equilibration period prior to the next sample run. Total run time per sample was 19.2 min, and all injection volumes were 10 and 20 µl on column.
Mass Spectrometric Conditions. Mass spectrometric analysis of 20-HETE formation was performed using a Thermo Finnigan MSQ single quadrupole mass spectrometer and a Thermo Finnigan TSQ Quantum Ultra triple quadrupole mass spectrometer, both operated in negative electrospray ionization (ESI) mode. Analysis was carried out on the MSQ with a probe temperature and voltage of 350°C and 3.0 kV, respectively. Cone voltage was set at 75 V, and selective ion monitoring was carried out for specific m/z 320.5 (20-HETE) and m/z 326.5 (20-HETE-d6, internal standard). Analysis was carried out on the TSQ operated in negative ESI selected reaction monitoring (SRM) mode with unit resolutions at both Q1 and Q3 set at 0.70 full width at half maximum. The SRM transitions of m/z 319.3 
245 (collision energy 15 eV, scan time 0.20 s) for 20-HETE and m/z 325.3 251 (collision energy 15 eV, scan time 0.20 s) for IS were monitored. Parameters were optimized to obtain the highest [M-H]+ ion abundance and were as follows: capillary temperature, 350°C; spray voltage, 3500 kV; and source collision-induced dissociation, 0 V. Sheath gas, auxiliary gas, and ion sweep gas pressures were set at 50, 20, and 0, respectively. Collision gas pressure was set at 1.2 mTorr.
Mass spectrometric analysis of HET0016 was performed using a Thermo Finnigan MSQ single quadrupole mass spectrometer and a Thermo Finnigan TSQ Quantum Ultra triple quadrupole mass spectrometer, both operated in positive ESI mode. Analysis was carried out on the MSQ (supplemented with a cone wash using 100% MeOH) with a probe temperature of 350°C and a needle voltage of 3.8 kV. Cone voltage was set at 63 V, and selective ion monitoring was carried out for specific m/z 207.15 (HET0016) and m/z 206.29 (IS). Detection of HET0016 in brain cortical tissue extracts was employed using the TSQ Quantum Ultra system operated in positive ESI SRM mode with unit resolutions at both Q1 and Q3 set at 0.70 full width at half maximum. The SRM transitions of m/z 207 130 (collision energy 31 eV, scan time 0.10 s) for HET0016 and m/z 206 108.1 (collision energy 21 eV, scan time 0.10 s) for IS were monitored. Capillary temperature was set at 350°C, spray voltage was set at 4200 kV, and source collision-induced dissociation was set at 0 V. Sheath gas, auxiliary gas, and ion sweep gas pressures were set at 50, 20, and 5, respectively. Collision gas pressure was set at 1.2 mTorr. Analytical data were acquired and analyzed using XCaliber software version 1.4 (Thermo Fisher Scientific).
Pharmacokinetic Parameter Determination and Statistical Methods. HET0016 pharmacokinetic parameter estimates were determined by noncompartmental methods using WinNonlin version 4.1 (Pharsight, Mountain View, CA). An unpaired Student's t test was performed for comparisons between two groups, whereas a one-way analysis of variance was performed for comparisons among three or more groups, with individual differences determined via a Dunnett's multiple comparison post hoc. Values that are statistically significant are designated by asterisks (*, p < 0.05; **, p < 0.01). Statistical analyses were performed using GraphPad Prism 2004 (GraphPad Software, Inc., San Diego, CA).
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Results |
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Mechanism of HET0016 Inhibition of 20-HETE Formation. Formation of 20-HETE in incubations containing rat brain cortical microsomes (325 µg of total protein), NADPH (1 µM), and arachidonic acid (0.2-70 µM) in the presence and absence of HET0016 (24 nM) were determined. As depicted in Fig. 2A, maximal 20-HETE formation rate in vehicle control microsomes was 10.2 pmol/mg/min with saturation observed at arachidonic acid concentrations above 30 µM. Coincubation with 24 nM HET0016 reduced the maximal formation rate to 0.69 pmol/mg/min. Inhibition of 20-HETE formation in rat brain cortical microsomes persisted despite increasing concentrations of arachidonic acid. Concentrations up to 120 µM arachidonic acid incubated in the presence of 24 nM HET0016 did not alter the formation rate, thereby demonstrating noncompetitive inhibition by HET00016.
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HET0016 Inhibition of 20-HETE Formation. Brain cortical microsomes were incubated with arachidonic acid in the presence or absence of HET0016 to determine the inhibition of 20-HETE formation in microsomes. Figure 3A demonstrates that rat brain cortical microsomal formation rate was inhibited by HET0016 with significant inhibition at 30 nM (1.81 ± 1.14 pmol/mg/min) and 90 nM (0.92 ± 0.02 pmol/mg/min) concentrations as compared with control (6.46 ± 2.5 pmol/mg/min). In addition, rats were treated in vivo with 10 mg/kg HET0016 or lecithin vehicle followed by sacrifice and tissue harvesting 1 h after dosing. Brain cortical microsomes were harvested for ex vivo evaluation of 20-HETE microsomal formation in the absence of inhibitor. Figure 3B demonstrates the 20-HETE formation rate in microsomes obtained from treated and untreated animals.
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Brain 20-HETE Formation after Intravenous Administration of HET0016. A total of 40 rats were serially sacrificed for measurement of 20-HETE concentration in cortical brain tissue at 0, 5, 10, 30, 60, 180, 360, and 1440 min after HET0016 single i.v. dose administration (n = 5 per time point). As depicted in Fig. 6, tissue concentrations of 20-HETE were decreased within 5 minutes of dose administration and remained significantly reduced for 60 min. Consequently, the 20-HETE concentration partially recovered to 70% at 180 min and returned to control levels by 24 h. In addition, the brain to plasma ratio of HET0016 was also determined at the 5, 10, 30, and 60 min time points after HET0016 administration. The HET0016 brain cortical tissue to plasma ratio was 7.5 ± 1.1 at 5 min, 9.5 ± 1.6 at 10 min, 2.9 ± 0.5 at 30 min, and 2.2 ± 0.3 at 60 min. The time course of cortical tissue and plasma concentrations in these animals is presented in Fig. 7.
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Discussion |
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Our conclusion that HET0016 is a selective, noncompetitive inhibitor of 20-HETE formation in rat brain microsomes is based on the observation that HET0016 (24 nM) reduced the maximum velocity of 20-HETE formation in vitro that was unaltered by increasing arachidonic acid concentrations. These data are consistent with a previous report by Seki et al. (2005), who demonstrated that HET0016 at concentrations of 20 and 40 nM reduced Vmax without altering Km in cell membranes from a recombinant CYP4A1 expression system, thereby implying noncompetitive inhibition. In a study by Miyata et al. (2001), EET metabolites were combined to determine the effect of the inhibitor on 20-HETE versus the sum total of EET peaks observed in the kidney microsomal systems. This study demonstrated that HET0016 has a selectivity window for 20-HETE inhibition at 10-6 to 10-7 M concentrations, whereas the mechanism of HET0016 inhibition in kidney microsomes was not determined in this study. These results are also consistent with previous studies by our laboratory that have demonstrated that HET0016 specifically inhibits microsomal formation of 20-HETE as compared with individual EETs (Poloyac et al., 2006).
Prior studies have suggested that, although HET0016 is highly distributed and is a very potent inhibitor of 20-HETE formation, its use is limited by its poor aqueous solubility at neutral pH and the instability of HET0016 at acidic pH (Nakamura et al., 2003). This led to studies exploring new pyrazole and isoxazole derivatives of HET0016 as new chemical inhibitors of this pathway. In the present study, we demonstrated that a hydroxypropyl-β-cyclodextrin formulation of HET0016 greatly increases the aqueous solubility of this compound and thereby creates a useful intravenous form. Furthermore, we demonstrated that this formulation of HET0016 produces a reproducible plasma profile and exhibited rapid brain disposition and reductions in 20 HETE within 5 min of administration. This observation is consistent with multiple other studies that have successfully used HPβCD to increase the aqueous solubility of small molecules for intravenous administration (Jarho et al., 1995; Trapani et al., 1998; Govindarajan and Nagarsenker, 2005). Furthermore, the concentrations of 15% HPβCD administered as a 1-ml solution intravenously have been used previously without adverse effects in animal models and humans (Pitha et al., 1988; Gould and Scott, 2005). Collectively, these data suggest that a formulation of HET0016 with HPβCD is a viable method for in vivo inhibition of 20-HETE in the rat brain, thereby facilitating future studies to determine the role of 20-HETE in the pathogenesis of neurovascular disease.
Solid phase extraction with HPLC MS/MS detection of HET0016 was demonstrated as a reproducible method for the quantification of tissue concentrations. This extraction method is the same method previously reported by our laboratory for the quantification of 20-HETE (Poloyac et al., 2005). Although these methods differ in mass spectrometric detection conditions, quantification of 20-HETE and HET0016 can be completed via sequential injections of a single tissue extract by this method.
The pharmacokinetic parameter estimates demonstrate that HET0016 has a short biological half-life. Previously, our laboratory determined pharmacokinetic parameter estimates after intraperitoneal administration of a 10 mg/kg dose of HET0016 dissolved in lecithin (Poloyac et al., 2006). Brain concentrations were less than 10% of plasma concentrations at 1 h after intraperitoneal dosing in our previous study as compared with 220% at 1 h after HET0016/HPβCD intravenous dosing in the present study. Although this is not a direct comparison of dosage routes and formulations, we can state that lower doses of intravenous HET0016/HPβCD are required for brain inhibition of 20-HETE potentially due to greater brain penetration; however, continuous infusion is likely to be necessary to maintain this inhibition in the brain due to the short biological half-life of HET0016 in vivo.
In summary, the results of this study provide evidence that HET0016 is a noncompetitive inhibitor of brain cortical 20-HETE formation. We also demonstrate that, despite its low intrinsic aqueous solubility, HET0016 can be formulated with HPβCD to increase aqueous solubility for intravenous administration. Furthermore, we demonstrated that HET0016 produces a very rapid, short duration inhibition of 20-HETE formation after single dose administration. These data will allow for future studies to elucidate the in vivo pharmacology and mechanism of inhibition by HET0016 and the role of 20-HETE in diseases of neurovascular origin.
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Footnotes |
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Y.M. and M.M.K. contributed equally to this work.
ABBREVIATIONS: HETE, hydroxyeicosatetraenoic acid; EET, epoxyeicosatrienoic acid; P450, cytochrome P450; 17-ODYA, 17-octadecynoic acid; DDMS, N-methylsulfonyl-12,12-dibromododec-11-enamide; HET0016, N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine; TS-011, N-(3-chloro-4-morpholin-4-yl) phenyl-N'hydroxyimidoformamide; HPβCD, hydroxypropyl-β-cyclodextrin; ddH2O, distilled, deionized water; QC, quality control; MS/MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; IS, internal standard; ESI, electrospray ionization; SRM, selected reaction monitoring.
Address correspondence to: Dr. Samuel M. Poloyac, 807 Salk Hall, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261. E-mail: poloyac{at}pitt.edu
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70% reduction in 20-HETE formation rate. B, rats were treated with HET0016 (10 mg/kg) or lecithin control intraperitoneally and were sacrificed 1 h later for measurement of 20-HETE formation in microsomes ex vivo. Microsomes were isolated from HET0016 and vehicle-treated animals 1 h after dosing. In vitro 20-HETE formation was then assessed in the absence of inhibitor. This figure depicts the persistent inhibition of 20-HETE formation in animals treated with HET0016. Statistically significant differences are denoted by asterisks; **, p < 0.01.
