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.
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
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