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Pharmacokinetics and metabolism of the reactive oxygen scavenger, ⁺-phenyl-N-tert-butyl nitrone (PBN) and the effects of 3-nitropropionic acid in the Sprague-Dawley rat / Mary E. Trudeau-Lame

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Identifier	Thesis 1713
Author	Trudeau-Lame, Mary E
Title	Pharmacokinetics and metabolism of the reactive oxygen scavenger, ⁺-phenyl-N-tert-butyl nitrone (PBN) and the effects of 3-nitropropionic acid in the Sprague-Dawley rat / Mary E. Trudeau-Lame
Publisher	Central Connecticut State University
Date	2002
Resource Type	Master's Thesis
Notes	Animal models of Huntington's disease (HD) that utilize 3-nitropropionic acid (3-NPA) in the initiation of oxidative stress and accelerated brain-aging have been extensively studied. When administered to rodents, 3-NPA irreversibly inhibits succinate dehydrogenase which results in the initiation of oxidative stress and the subsequent formation of selective lesions in the striatum, resembling the neuropathology associated with HD. 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. Free radical spin traps are non-paramagnetic species that react with reactive free radicals to produce stable free radicals, thus blocking the free radicals from damaging cellular components. With the suggested role of oxidative stress in 3-NPA toxicity, many free radical spin traps have been investigated as to their effectiveness in attenuating 3-NPA neurodegeneration. An example of a spin trap that is widely used in biochemical studies to trap free radicals or reactive oxygen species (ROS) is the nitrone derivative ·-phenyl N-tert butylnitrone (PBN). PBN’s pharmacological effects in animal models are extensive ranging from protection against lethal endotoxic shock and from brain injury in experimental models of stroke. Furthermore, chronic low-level administration of PBN to aged rats reverses their age-enhanced susceptibility to stroke even several days after the last dose. 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 p38· MAP kinase, NF-kappaB, inducible nitric oxide synthase, and cyclooxygenase-2. Besides PBN, alternate spin traps including 5,5-dimethyl-1-pyrrolin-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N-oxide (DEPMPO) have also been investigated as potential neuroprotectants. Despite its role as a neuroprotectant in many rodent models of stroke and aging, PBN exacerbates, rather than attenuates 3- NPA mediated neurotoxicity in the HD model. A possible explanation for this behavior has been attributed to its inhibitory effect on the metabolic pathways of 3-NPA, which results in increased concentration of the neurotoxin in the brain. Alternately, it is possible that 3-NPA functions as a stimulant of PBN metabolism, resulting in decreased levels of the neuroprotectant in the brain. Despite the extensive attention that PBN has received from a medicinal chemistry/ pharmacology perspective, in vivo pharmacokinetics/metabolism studies on the nitrone remains relatively scarce, 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. Therefore, a major theme of this thesis work was to investigate and 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 towards establishing an in vitro-in vivo correlation for PBN clearance and for the purposes of characterizing its major metabolic pathways. In addition, the possibility that 3-NPA acts as a stimulant of PBN metabolism in the rat was also examined by in vitro and in vivo experiments. Plasma concentrations after intravenous (i.v.) administration of PBN (10mg/kg) declined mono-exponentially with a terminal half-life of 2.01 ± 0.35 hours 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 excellent agreement with the hepatic clearance (CLh) value (11.5 ml/min/kg) predicted from in vitro liver microsomal data suggesting that PBN clearance (CLp) in rats was entirely due to hepatic metabolism. The peak plasma concentration (Cmax) following oral (p.o.) (20 mg/kg) and subcutaneous (s.c.) (30 mg/kg) PBN administration was 7.35 ± 1.92 and 3.56 ± 0.66 ·g/ml, whereas the area under the curve estimated to infinity (AUC(0-¥) ) was 23.89 ± 5.84 and 15.96 ± 3.10 ·g-hr/ml, respectively. Mean oral bioavailability was 2.2-fold higher after p.o. administration when compared with the s.c. route. When 3-NPA (30 mg/kg) was co-administered with PBN (10 mg/kg) intravenously, the mean plasma clearance of PBN increased ~ 5-fold to 63.48 ± 14.96 ml/min/kg. This observation suggested that 3-NPA stimulated PBN clearance in vivo. Stimulation of PBN metabolism by 3-NPA was also discernible in male rat liver microsomes. For instance, the predicted hepatic clearance of PBN in the absence or the presence of 3-NPA (1 ·M) was 11.5 ml/min/kg and 41.51 ml/min/kg, respectively. Interestingly, the predicted in vitro PBN clearance in the presence of 3-NPA was within 2-fold of the actual clearance of PBN in the presence of 3-NPA. Studies on the metabolism of PBN in male rat microsomes indicated that metabolism was dependent on NADPH suggesting the principal involvement of P450 in the metabolism of the spin trap. Biotransformation studies in liver microsomes indicated the formation of a single metabolite M1 with a molecular mass 16 mass units higher than PBN suggesting that P450 mediated monohydroxylation of PBN was its principal metabolic fate. Subsequent studies using heterologously expressed rat P450 isozymes indicated the exclusive involvement of P450 2C11 in the monohydroxylation 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 ·-hydroxyphenyl-N-tert-butyl nitrone (4-HOPBN). The structure of M1 was unambiguously established as 4-HOPBN on the basis of: (a) obvious LC Rt differences between M1 and the authentic hydroxamate standard, (b) 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 (c) comparison of the LC/MS/MS properties with a 4- HOPBN standard (LC retention times, and MS CID fragmentation patterns). Comparative studies in human liver microsomes also indicated the potential low clearance of PBN in humans. Furthermore, metabolism of PBN by human liver microsomes also led to the formation of 4-HOPBN, the formation of which was mediated by P450 2C19 and 1A2. Based on the results obtained from this thesis, it is speculated that 4-HOPBN is an “active” PBN metabolite that provides an additive effect to the pharmacological action of PBN in vivo. Furthermore, the in vitro and in vivo stimulation of PBN metabolism by 3-NPA (via interactions with P450 2C11, the principal P450 isozyme, involved in the metabolism of PBN) provides a rationale for the lack of neuroprotective properties of this spin trap in the 3-NPA model of HD.
Subject	Free radicals (Chemistry)
Department	Department of Chemistry and Biochemistry
Advisor	LaFontaine, Michael, 1970-
Type	Text
Digital Format	application/pdf
Language	eng
OCLC number	52412855
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Pharmacokinetics and metabolism of the reactive oxygen scavenger, ⁺-phenyl-N-tert-butyl nitrone (PBN) and the effects of 3-nitropropionic acid in the Sprague-Dawley rat / Mary E. Trudeau-Lame

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