Abstract
The pharmacokinetics of the spin-trap α-phenyl-N-tert-butylnitrone (PBN) was investigated in male Sprague-Dawley rats. Plasma concentrations after i.v. administration (10 mg/kg) declined monoexponentially with a terminal half-life of 2.01 ± 0.35 h and total plasma clearance (CLp) and volume of distribution at steady state (Vdss) averaged 12.37 ± 3.82 ml/min/kg and 1.74 ± 0.5 l/kg, respectively. The observed CLp was in close agreement with the blood clearance (CLb) value (11.5 ml/min/kg) predicted from in vitro liver microsomal incubations suggesting that PBN CLp in rats is predominantly due to hepatic metabolism. Peak plasma concentration (Cmax) following p.o. (20 mg/kg) and s.c. (30 mg/kg) PBN administration was 7.35 ± 1.92 and 3.56 ± 0.66 μg/ml, whereas the area under the concentration-time curve from 0 to infinity was 23.89 ± 5.84 and 15.96 ± 3.10 μg-h/ml, respectively. The mean oral bioavailability of PBN was 85.63 ± 20.93%. Biotransformation studies indicated the P450 2C11-catalyzed hydroxylation of PBN to M1. Potential sites of hydroxylation included the benzylic carbon resulting in phenyl-N-tert-butylhydroxamic acid or the phenyl ring that would afford α-hydroxyphenyl-N-tert-butylnitrone (HOPBN). The structure of M1 was established as α-4-Hydroxyphenyl-N-tert-butylnitrone (4-HOPBN) on the basis of: 1) obvious LC Rtdifferences between M1 and the authentic hydroxamate standard, 2) P450 catalyzed hydroxylation of [2H]PBN that contained a deuterium instead of a hydrogen atom on its benzylic position and which afforded [2H]M1, and 3) comparison of the liquid chromatography-tandem mass spectrometry properties with a synthetic 4-HOPBN standard. We speculate that 4-HOPBN is an “active” PBN metabolite that provides an additive effect to the pharmacological action of PBN in vivo.
Radical-mediated oxidative tissue damage is often times presumed to be the rate-limiting step in the initiation of neurodegenerative processes including stroke. Consequently, free radical spin traps or reactive oxygen scavengers (antioxidants) such as nitrones or phenolic compounds have received considerable attention as potential neuroprotectants in age-associated oxidative tissue damage and as anti-inflammatory agents (Kotake, 1999). α-Phenyl-N-tert-butylnitrone (PBN1) (Fig. 1) constitutes the parent compound of the nitrone family of spin-trapping agents commonly used to trap free radicals. PBN is known to react with short-lived oxygen- or carbon-centered radicals at the nitrone double bond resulting in a relatively stable nitroxide radical, which can no longer propagate destructive radical chain reactions. Furthermore, the nitroxide radical is stable enough to diffuse from the site of its generation, thereby preventing concentrated, debilitating tissue damage. The neuroprotective effects of PBN in animal models are quite extensive (Floyd et al., 2002). For instance, chronic low-level administration of PBN to aged rats reverses their age-enhanced susceptibility to stroke even several days after the last dose (Hensley and Floyd, 2002). Besides its role as a neuroprotectant, PBN also functions as a potent anti-inflammatory agent by virtue of its inhibitory effects on the activation/mRNA synthesis of inflammatory mediators such as p38α mitogen-activated protein kinase, nuclear factor-κB, inducible nitric oxide synthase, and cyclooxygenase-2 (Kotake et al., 1998). The pharmacological basis for the neuroprotective, antiaging and anti-inflammatory effects of PBN has been attributed to its radical-trapping properties and has also prompted medicinal chemistry efforts designed to increase the reactivity of PBN toward free radicals (Fevig et al., 1996; Thomas et al., 1996).
Despite the extensive attention that PBN has received from a medicinal chemistry/pharmacology perspective, in vivo pharmacokinetics/metabolism studies on the nitrone remain relatively scarce (Chen et al., 1990), particularly in the rat, the animal model of choice for the characterization of PBN pharmacology. As a consequence, the pharmacokinetic-pharmacodynamic relationships for its mechanism of action remain unclear. For example, comparison of the in vitro antioxidant properties of PBN and butylated hydroxy toluene (BHT) in rat lipid peroxidation models indicates that BHT is ∼1000-fold more effective to PBN as an antioxidant, however, PBN demonstrates greater in vivo antioxidant activity (Carney et al., 1991; Floyd, 1996). This outcome may be associated with the superior rat pharmacokinetic properties of PBN when compared with BHT. Furthermore, unlike the spin trapping agents 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide that protect against 3-nitropropionic acid (3-NPA)-induced neurodegeneration in a rodent model of Huntington's disease (LaFontaine et al., 2000), PBN reportedly exacerbates 3-NPA toxicity (Prasad et al., 1998). Although, this phenomenon has been attributed to the inhibitory effects of PBN on the metabolic pathways of 3-NPA (Butterfield et al., 2001), stimulation of PBN metabolism by 3-NPA that increases the overall clearance of the spin trap in rodents may provide an alternate explanation (Butterfield et al., 2001). Thus, in contrast to earlier hypothesis on the free radical trapping activity as the sole criterion for the pharmacological action of PBN, its pharmacokinetic properties (such as clearance, half-life, and bioavailability) may also be of equal importance (Floyd et al., 2002). Therefore, the goal of these studies was to examine the pharmacokinetics, bioavailability, and the metabolism of PBN after single oral, intravenous, and subcutaneous doses in male Sprague-Dawley rats. In vitro metabolism studies were also undertaken in male rat liver microsomes toward establishing an in vitro-in vivo correlation for PBN clearance and for the purposes of characterizing its major metabolic pathways.
Experimental Procedures
Materials.
PBN was purchased from Sigma-Aldrich (St. Louis, MO). [2H]Benzaldehyde was purchased from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals and reagents were obtained from Aldrich Chemical Co. 1H NMR spectra in dimethyl sulfoxide-d6 or CDCl3 were recorded on a Varian Unity M-400 MHz spectrometer; chemical shifts are expressed in parts per million (ppm, δ), and spin multiplicities are given as s (singlet), d (doublet), and m (multiplet). Rat liver microsomes were generated at Pfizer using liver tissue from male Sprague-Dawley rats. Recombinant rat P450 isozymes were obtained from BD Gentest Corp. (Woburn, MA). α-4-Hydroxyphenyl-N-tert-butylnitrone (4-HOPBN) was synthesized in ∼10% yield by an acid-catalyzed condensation of 4-hydroxybenzaldehyde and N-tert-butylhydroxyl amine according to a previously published method (Hinton and Janzen, 1992).
[2H]α-Phenyl-N-tert-butylnitrone.
A mixture containing glacial acetic acid (3.7 ml, 60 mmol), benzaldehyde containing a deuterium on its benzylic carbon ([2H]benzaldehyde, 1.07 g, 10 mmol), 2-methyl-2-nitropropane (2.06 g, 20 mmol), and activated zinc (1.96 g, 30 mmol) in 95% ethanol (100 ml) was stirred for 4 h and then stored in a refrigerator overnight. Upon evaporation of the solvent, the residue was dissolved in diethyl ether (50 ml), extracted with water, 1 N sodium bicarbonate, and then again with water (50 ml each). The ether extract was dried (MgSO4), filtered, and concentrated. The crude product was recrystallized from hexanes to afford a white crystalline solid (1.5 g, 84%).1H NMR (dimethyl sulfoxide-d6) δ 8.32–8.35 (m, 2H, ArH), 7.37–7.39 (m, 3H, ArH), 1.48 (s, 9H, tert-butyl). Deuterium enrichment was 100% based on the complete absence of the resonance at δ 7.82 ppm attributed to the benzylic proton in PBN. Liquid chromatography-tandem mass spectrometry LC/MS/MS analysis revealed a single peak (Rt = 11.54 min) with a protonated molecular ion (MH+) at 179 (one mass unit higher than PBN) and a base fragment at 123.
Phenyl-N-tert-butylhydroxamic acid was prepared via a slight modification of a previously published method (Bordwell and Liu, 1996). Benzoyl chloride (2.4 ml, 20 mmol) was added dropwise to N-tert-butylhydroxyl amine · HCl salt (1.1 g, 10 mmol) and triethyl amine (5.6 ml, 40 mmol) in anhydrous acetonitrile (20 ml) at 0°C under nitrogen. The resulting mixture was stirred overnight and then mixed with water (40 ml) and extracted with methylene chloride (2 × 20 ml). The organic layer was dried (MgSO4), filtered, and concentrated. The residue was dissolved in 20 ml of 1 N ethanolic NaOH solution and stirred for 1 h. The aqueous solution was extracted once with methylene chloride (10 ml) and then neutralized with 1 N HCl and extracted with diethyl ether (2 × 25 ml). The combined ether extracts were dried (MgSO4), filtered, and concentrated. The crude hydroxamic acid was recrystallized from ethyl acetate/hexanes (5:95) to afford the desired product as a light brown solid (1.4 g, 70%). Melting point 112 to 113°C (Lit melting point = 110 to 113°C;Bordwell and Liu, 1996). 1H NMR (CDCl3) δ 8.10–8.12 (d, 1H, J= 8 Hz, OH), 7.37–7.63 (complex m, 5H, ArH), 1.32 (s, 9H,tert-butyl). LC/MS/MS analysis revealed a single peak (Rt = 12.8 min) with a protonated molecular ion (MH+) at 194 and a base fragment at 138.
Microsomal Incubations.
PBN half-life in microsomes was determined in triplicate following its incubation (1 μM) with rat liver microsomes (pool of 10-∼15 livers from male Sprague-Dawley rats, P450 concentration = 0.25 μM) in 0.1 M potassium phosphate buffer (pH = 7.4) at 37°C. The reaction mixture was prewarmed at 37°C for 2 min before adding NADPH (1.2 mM). The final incubation volume was 600 μl. Aliquots (75 μl) of the reaction mixture at t = 0, 5, 15, and 30 min (time period associated with reaction linearity) were added to acetonitrile (150 μl) containing midazolam (0.085 μg/ml) as internal standard and the samples were centrifuged at 2500gfor 5 min prior to LC/MS/MS analysis. For metabolite identification studies, the concentration of PBN was raised to 100 μM. For control experiments, NADPH was omitted from these incubations. The scaling-up of the microsomal t1/2 data to estimate the rat hepatic intrinsic clearance (CL′int) and subsequently the blood clearance (CLb) was conducted according to our previously published protocol (Kuperman et al., 2001). Briefly, the scaling-up of the t1/2 data in liver microsomes, reflecting PBN depletion, was performed using the following equation (Houston, 1994; Obach et al., 1997): For rats, the liver and body weights were 40 g/kg body weight and 0.25 kg, respectively, and the microsomal protein concentration was 0.39 mg/ml. The CLb was estimated using the nonrestricted well stirred model (eq. 1) (Pang and Rowland, 1977). The blood to plasma ratio of PBN in the rat at 1 and 10 μM was 0.97 (n = 3) and 1.02 (n = 3), respectively.
Measurement of the Blood to Plasma Ratio for PBN.
Fresh blood was obtained in heparinized containers from Sprague-Dawley rats. A portion of the blood was centrifuged (3000g for 5 min) to harvest plasma. Aliquots of rat blood or plasma (4 ml) were incubated with PBN (1 and 10 μM) at 37°C for 25 min (pH = 7.4) in a shaking water bath (∼100 rpm). The blood samples fortified with PBN were centrifuged at 3000g for 5 min to harvest plasma. The plasma samples (harvested from blood and those initially fortified with PBN) were treated with 2 volumes of ice-cold acetonitrile containing midazolam as internal standard, centrifuged (2500g, 5 min) prior to LC/MS/MS analysis. All incubations were conducted in triplicate.
Metabolism by Heterologously Expressed P450 Isoforms.
PBN (100 μM) was incubated with microsomes from cells containing rat recombinant P450s 3A1, 3A2, 2C11, 2C12, and 2C13 (P450 concentration = 0.05 μM) in the presence of NADPH (1.2 mM) in 0.1 M potassium phosphate buffer (pH = 7.4) at 37°C for 30 min. Reactions were terminated by addition of ice-cold acetonitrile (2:1, v/v). The formation of 4-HOPBN was assessed by LC/MS/MS.
LC/MS/MS Assay for PBN Quantitation.
PBN depletion was monitored on a Sciex API model 3000 LC/MS/MS triple quadrupole mass spectrometer or a Micromass Quattro Ultima (Micromass Inc., Beverly, MA). Analytes were chromatographically separated using a Hewlett Packard Series 1100 HPLC system [Phenomenex Primesphere 5 μ C18-HC 30 × 2.0 mm column (Phenomenex, Torrance, CA) using a 2- and 3-min binary gradient consisting of a mixture of 95% water/5% acetonitrile with 0.1% acetic acid (Solvent A) and 95% acetonitrile/5% water with 0.1% acetic acid (Solvent B) and a flow rate of 1.0–1.5 ml/min]. Ionization was conducted in the positive ion mode at the ionspray interface temperature of 400°C (LC/MS/MS) or 100°C (Micromass), and nitrogen was used as a nebulizing and heating gas. PBN and midazolam were analyzed using multiple reaction monitoring at mass rangesm/z 178 → 122 and 326 → 291, respectively. In the case of PBN, this reaction follows the protonated parent mass MH+ = 178 to its corresponding collision-induced dissociated fragment at m/z 122, which corresponds to the loss of the tert-butyl group on PBN. The dynamic range of the assay was 0.005 to 20.0 μg/ml.
LC/MS/MS Assay for Metabolite Identification.
Qualitative assessments of PBN metabolism were conducted on a Sciex API model 2000 LC/MS/MS triple quadrupole mass spectrometer (Sciex, Thornhill, Ontario, Canada) in conjunction with an LDC Analytical SpectroMonitor 3200 variable wavelength UV-detector. PBN and its metabolites were chromatographically separated using a Hewlett Packard Series 1100 HPLC system [Zorbax Eclipse XDB-C84.6 × 150 mm column using a binary gradient consisting of a mixture of 10 mM ammonium formate, 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) at a flow rate of 1 ml/min]. The LC gradient was programmed as follows: solvent A to solvent B ratio was held at 100:0 (v/v) for 3 min and then adjusted from 100:0 (v/v) to 10:90 (v/v) for 20 min and from 10:90 (v/v) to 100:0 (v/v) from 20 to 25 min. Postcolumn flow was split such that mobile phase was introduced into the mass spectrometer via an ion spray interface at a rate of 50 μl/min. The remaining flow was diverted to the UV detector positioned in line so as to provide simultaneous UV detection at λ = 254 nm and total ion chromatogram. Ionization was conducted in the positive ion mode at the ionspray interface temperature of 150°C using nitrogen as a nebulizing gas. Ion spray voltage was 4.5 kV, and the orifice voltage was 30 eV. Initial Q1 scans were performed betweenm/z 50 to 500. Potential PBN metabolites in rat liver microsomes were identified by comparing t = 0 samples to t = 30 min samples (with or without NADPH), and structural information was generated from collision-induced dissociation of the corresponding protonated molecular ions.
Animal Studies.
All procedures were approved by the Pfizer Institutional Animal Care and Use Committee. Fasted male Sprague-Dawley rats (∼200–240 g,n = 4 for each route of administration) with jugular vein catheters (Charles River Labs, Wilmington, MA) were administered PBN in phosphate-buffered saline as an i.v. (10 mg/kg), p.o. (20 mg/kg), or s.c. (30 mg/kg) bolus solution. Rats received food (standard rodent diet) at 4 h following administration and were allowed full access to water throughout the study. Blood samples (400 μl) were collected from the jugular vein at appropriate time intervals in heparinized tubes, centrifuged (5 min at 3000g) to harvest plasma. Plasma was thawed, and 100 μl was diluted with 2 volumes of acetonitrile containing midazolam as internal standard. The samples were centrifuged, and the supernatant was injected for LC/MS/MS analysis as described above. Urine samples (0–24 and 24–48 h) were also collected from rats after i.v. and p.o. dosing with PBN and stored at 0°C until analysis. Aliquots (100 μl) of the urine were diluted with 3 volumes of acetonitrile containing midazolam as internal standard. The samples were centrifuged (5 min at 3000g) and the supernatant was evaporated at 37°C under a steady stream of nitrogen gas. The residue was reconstituted with mobile phase (100 μl) and analyzed for unchanged PBN by LC/MS/MS as previously described.
Data Analysis.
Plasma concentration-time profiles were analyzed using the well established noncompartmental method in WinNonLin v2.1 (Pharsight, Mountain View, CA). The peak plasma concentration (Cmax) observed after oral dosing and the time at which it was observed (Tmax) were determined by direct inspection of the individual plasma concentration-time profiles. The terminal slope of the ln(concentration) versus time plot was calculated by linear least-squares regression, and the half-life was calculated as 0.693 divided by the absolute value of the slope. The area under the plasma concentration-time curve from time 0 to infinity (AUC0-∞) was calculated by the linear trapezoid rule. The total plasma clearance (CLp) of i.v. PBN was calculated as the i.v. dose divided by the AUC0-∞ of i.v. PBN and the volume of distribution at steady state (Vdss) by the method of Benet and Galeazzi (1979). The relative bioavailability (F) of the oral doses was calculated by using the following equation:
Results and Discussion
Figure 2 displays the mean plasma PBN concentration-time profile in male Sprague-Dawley rats after administration of i.v., p.o., and s.c. doses of PBN, and the resulting pharmacokinetic parameters are summarized in Table1. Plasma concentrations after i.v. PBN administration declined rapidly with a terminal half-life of 2.01 ± 0.35 h. CLp and Vdss averaged 12.37 ± 3.82 ml/min/kg and 1.74 ± 0.5 l/kg, respectively. Scale-up of the CL′int reflecting PBN depletion in rat liver microsomes resulted in a projected CLb of 11.5 ml/min/kg that correlated reasonably well with the actual rat CLp (blood to plasma ratio of PBN was determined to be 1). Furthermore, urinary analysis after i.v. administration revealed that less than 0.5% of the administered dose was excreted as unchanged PBN after 48 h. Collectively, these results strongly suggest that hepatic metabolism constitutes the major clearance pathway of PBN in male Sprague-Dawley rats. The mean (±S.D.)Cmax of PBN observed after p.o. administration was 7.35 ± 1.92 μg/ml and occurred 0.56 ± 0.31 h (Tmax) after dosing, and the corresponding AUC0-∞ was 23.89 ± 5.84 μg-h/ml. The terminal half-life after oral administration (2.77 ± 0.55 h) was not significantly different (P > 0.1) from that observed after i.v. administration, suggesting that elimination, and not absorption, is the rate-limiting step in PBN disposition after p.o. dosing. The lack of statistical significance between the half-lives observed after i.v. and p.o. dosing also may reflect the relatively small (n = 4) number of animals used in the study. Overall, PBN demonstrated an excellent oral bioavailability in these animals (oral F = 85.63 ± 20.93%). After s.c. administration, peak PBN concentration as judged from the Cmax was 3.56 ± 0.66 μg/ml (Tmax = 0.81 ± 0.38 h), whereas the AUC0-∞ was 15.96 ± 3.10 μg-h/ml, resulting in a mean s.c. bioavailability of 38.13 ± 7.40%. The lower s.c. bioavailability may reflect a slower rate and extent of absorption following this route of administration as judged from the slightly delayedTmax.
Following the incubation of PBN (100 μM) in NADPH-supplemented rat liver microsomes, the formation of a single metabolite M1 of greater polarity than PBN was discernible (PBNRt = 12.2 min; M1Rt = 10.0 min) (Fig.3, panel A). Formation of M1 required NADPH and PBN incubations conducted in the presence of denatured (boiled) rat liver microsomes supplemented with cofactor did not generate M1 (data not shown). The LC/MS/MS spectrum of M1 displayed a protonated parent ion (MH+) at 194 (i.e., 16 mass units higher than those observed for PBN consistent with a monohydroxylated-PBN metabolite). The collision-induced dissociation (CID) spectrum of MH+ 194 from M1 showed fragment ions at m/z 138 (100%) and 121 (75%) [i.e., 16 mass units higher than the corresponding fragment ions 122 (100%) and 104 (45%) observed in the CID spectrum of PBN (see Fig. 1)]. Overall, the CID spectrum of M1 was consistent with either P450-mediated hydroxylation on the benzylic carbon in PBN leading to phenyl-N-tert-butylhydroxamic acid (structure A) or hydroxylation on the phenyl ring leading to hydroxyphenyl-N-tert-butylnitrone (HOPBN) (structure B) (see Fig. 1). The fragment ion atm/z 57 (30–40%) (tert-butyl cation), which was observed in PBN and M1, ruled out hydroxylation on thetert-butyl group.
The proposal that P450-mediated metabolism of PBN led to the formation of phenyl-N-tert-butylhydroxamic acid (structure A) was ruled out on the basis of the following experiments: 1) LC/MS/MS analysis of microsomal incubations with [2H]PBN containing a deuterium instead of a hydrogen atom on its benzylic position (see Fig. 1) that revealed the formation of [2H]M1 (Rt = 10.0 min; Fig. 3, panel B). The CID spectrum of [2H]M1 revealed fragment ions one mass unit higher than those observed with M1 generated from microsomal incubations with PBN suggesting that the benzylic H/D atom(s) in PBN or [2H]PBN were intact (see Fig.1). 2) Obvious difference in the LC Rtof M1 (Rt = 9.62 min) and the synthetic standard of phenyl-N-tert-butylhydroxamic acid (Rt = 12.8 min) (Fig. 3, panel C), despite similar CID spectrums (see Fig. 1). Based on these observations, the only possible site of hydroxylation in PBN was on its phenyl ring. Since many P450-mediated hydroxylations on monosubstituted phenyl rings occur in the para position (Jones et al., 2002), we synthesized the corresponding 4-HOPBN derivative as the putative PBN metabolite M1. The mass spectrum as well as the LC retention time of synthetic 4-HOPBN was identical to that of the P450 generated M1 (Fig. 3, panel D) strongly suggesting that hydroxylation occurred on the para position on the aromatic ring in PBN (see Fig. 1). Qualitative analysis of rat urine also revealed the formation of HOPBN along with its correspondingO-glucuronide conjugate (MH+ = 370;Rt = 8.4 min). Overall, these results are also consistent with the previous report on the tentative identification of 4-HOPBN as the major PBN metabolite in rat liver microsomes (Reinke et al., 2000).
Qualitative analysis of the rat P450 isozyme responsible for PBN metabolism to 4-HOPBN was also conducted by incubating PBN (100 μM) with various baculovirus-insect cell-expressed rat P450 isozymes including P450 1A1, 1A2, 2B1, 3A1, 3A2, 2C11, 2C12, and 2C13. Experimental results (data not shown) indicated that the exclusive involvement of P450 2C11 in the oxidation of PBN to HOPBN. In contrast to our result on the involvement of the constitutively expressed and male predominant P450 2C11 in PBN metabolism, a previous report has described a significant reduction in [14C]PBN concentrations in the plasma and urine of rats pretreated with P450 2B1 and 1A1 inducers phenobarbital and 3-methylcholanthrene, respectively (Chen et al., 1991). A possible explanation for that accounts for these observations is that P450 2C11 may function principally in the metabolism of PBN at the physiological state whereas P450 2B1 and 1A1 contribution to PBN metabolism could be limited to rats pretreated with their respective inducers. For instance, in studies on the metabolism of the monoterpenoid limonene in male rats, P450 2C11 was implicated to play a major role in the metabolism of limonene in normal animals, whereas P450 2B1 involvement was evident in rats pretreated with phenobarbital (Miyazawa et al., 2002). Furthermore, in the case of 3-methylcholanthrene, it is well established that induction of P450 1A1 is also accompanied by transcriptional suppression of the P450 2C11 gene, which results in an overall decrease in the catalytic activity of the enzyme (Lee and Riddick, 2000). Thus, it is possible that in the absence of functional P450 2C11, induced P450 1A1 contributes toward PBN metabolism.
In summary, details on the rat pharmacokinetic and metabolism properties of PBN should aid in providing possible explanations for some of the observed discrepancies in its in vitro and in vivo pharmacology. Based on our results, the superior in vivo pharmacology of PBN as an antioxidant in vivo is consistent with its excellent pharmacokinetic properties in the rat (low CLpand moderate half-life) when compared with BHT, which has an extremely short plasma half-life (12 min) and a overall CLp(200 ml/min/kg) that exceeds the hepatic blood flow in the rat (Verhagen et al., 1989). Furthermore, the free phenolic PBN metabolite HOPBN that is known to retain the antioxidant properties of the parent compound (Reinke et al., 2000) may provide a additive effect to the pharmacological effects of PBN in vivo, a feature that may not be possible with BHT. Whether the attractive pharmacokinetic properties of PBN in the rat are retained in higher preclinical species and human remains unclear at this time. Future directions include studies aimed at evaluating the effects of PBN on 3-NPA metabolism and vice versa, which may provide some insight into the lack of neuroprotective properties of PBN in the 3-NPA model of Huntington's disease.
Footnotes
- Abbreviations used are::
- PBN
- α-phenyl-N-tert-butylnitrone
- BHT
- butylated hydroxy toluene
- 3-NPA
- 3-nitropropionic acid
- P450
- cytochrome P450
- 4-HOPBN
- α-4-hydroxyphenyl-N-tert-butylnitrone
- LC/MS/MS
- liquid chromatography-tandem mass spectrometry
- CL′int
- intrinsic clearance
- CLb
- blood clearance
- AUC
- area under the plasma concentration-time curve
- CLp
- total plasma clearance
- Vdss
- volume of distribution at steady state
- F
- relative bioavailability
- Cmax
- peak plasma concentration
- Tmax
- time for maximal oral exposure
- Rt
- retention time
- CID
- collision-induced dissociation
- structure A
- phenyl-N-tert-butylhydroxamic acid
- structure B (HOPBN)
- hydroxyphenyl-N-tert-butylnitrone
- Received July 31, 2002.
- Accepted November 12, 2002.
- The American Society for Pharmacology and Experimental Therapeutics