Drug metabolism and disposition: the biological fate of chemicals
The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bi... more The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bivalent sulfur metabolites. Methylthio analogs of phenylethylene glycol, with the methylthio group in both possible positions, were found as urinary metabolites in both species. These compounds were present in more than trace amounts. The excretion of 2-hydroxy-1-methylthio-1-phenylethane amounted to about 7% of the administered dose in the guinea pig, and about 2% in the rat, in o-24 hr urine samples. The positional isomer 1-hydroxy-2-methylthio-1-phenylethane was excreted in lesser amounts in both species. Acidic urinary metabolites derived from glutathione conjugates are species dependent. In this study, the only products observed in the rat were the mercapturic acids expected as a result of reaction of the oxide with glutathione. In the guinea pig, the major bivalent sulfur acids were the corresponding mercaptoacetic acids. Other related metabolites included a mercaptolactic and a mer...
Canadian Journal of Physiology and Pharmacology, Jun 1, 1992
Incubation of glyceryl trinitrate (GTN) with 5% (w/v) rat brain homogenate (RBH) resulted in biot... more Incubation of glyceryl trinitrate (GTN) with 5% (w/v) rat brain homogenate (RBH) resulted in biotransformation of the organic nitrate vasodilator drug to a mixture of glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN). Heating of the RBH at 100 °C for 5 min and (or) pretreatment with 5 mM N-ethylmaleimide at 37 °C for 10 min demonstrated that about two-thirds of the GTN biotransformation activity was due to a sulfhydryl-dependent enzymatic process resulting in the predominant formation of 1,2-GDN, and that the remaining biotransformation activity was due to a sulfhydryl-dependent nonenzymatic process resulting in the selective formation of 1,3-GDN. In a preliminary experiment, nitric oxide formation was observed during the incubation of GTN with RBH under anaerobic conditions. These data support the idea that some of the therapeutic and adverse effects of GTN are mediated through its action in the central nervous system.Key words: glyceryl trinitrate, biotransformation, rat brain homogenate, sulfhydryl-dependent enzyme, nitric oxide formation.
Radioligand binding studies were conducted to test the hypothesis that specific receptor sites fo... more Radioligand binding studies were conducted to test the hypothesis that specific receptor sites for glyceryl trinitrate (GTN) exist in vascular smooth muscle. The radioligand, [3H]GTN (9.98 Ci/mmol), was incubated with whole homogenate or subcellular fractions (10,000 X g pellet, 100,000 X g pellet and 100,000 X g supernatant) of bovine pulmonary vein (BPV) at 4 degrees C, 37 degrees C or room temperature. After incubation for 5, 15, 30 or 60 min, unbound and bound ligand were estimated by physical separation and liquid scintillation spectrometry. Separation of bound from free ligand was accomplished by vacuum filtration, centrifugation, equilibrium dialysis, gel filtration or precipitation. No evidence of specific binding of [3H]GTN to BPV whole homogenate or any subcellular fraction was observed. The viability of the BPV preparations was verified by demonstrating specific binding of [3H]nitrendipine to the 100,000 X g pellet. To ensure that the ligand was present throughout the incubation procedure in sufficient concentration to detect specific binding, the ability of BPV whole homogenate and subcellular fractions to biotransform GTN to glyceryl dinitrate was measured. After 60-min incubation at 37 degrees C, greater than 95% of the GTN added was unaltered; at 90 min, more than 83% of the radioactivity was associated with GTN. Our data are interpreted as not supporting the concept of a GTN receptor in BPV.
We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured r... more We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured rat lung fibroblasts (RLF), porcine kidney epithelial (PK1), bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Biotransformation of 0.1 microM GTN was linear over 30 min and the percentage of glyceryl dinitrate (GDN)/10(6) cells for BAE, BASM, RLF and PK1 at 30 min was 3.1, 2.3, 5.8 and 21.7%, respectively. At low GTN concentration (0.01-0.1 microM) there was a highly selective formation of 1,2-GDN, whereas at higher GTN concentration (greater than 1 microM) this selectivity was lost. Cyclic GMP accumulation did not occur in BAE or BASM at any GTN concentration, whereas for RLF and PK1 it was highly correlated to the rate of GDN formation. Upon re-exposure to GTN after treatment of RLF or PK1 cells for 3 hr with 0.1 mM GTN, there was an almost complete loss of the cyclic GMP response, GTN biotransformation was attenuated markedly and the selective formation of 1,2-GDN at low GTN concentration was absent. However, when GTN-treated cells were incubated for 18 hr in GTN-free media, there was a recovery of the cyclic GMP response, GTN biotransformation and selective 1,2-GDN formation toward control values.(ABSTRACT TRUNCATED AT 250 WORDS)
Studies on the physiological role of heme oxygenase (HO) require an inhibitor that will selective... more Studies on the physiological role of heme oxygenase (HO) require an inhibitor that will selectively inhibit HO activity without inhibiting the activity of either nitric oxide synthase (NOS) or soluble guanylyl cyclase (sGC). The objective of this study was to test a series of metalloporphyrins that have previously been shown to inhibit HO activity, for their ability to inhibit HO without inhibiting NOS or sGC activities. Measurement of activity of HO in rat brain microsomes and NOS in rat brain cytosol was made for samples incubated with metalloporphyrins (0.15-50 microM), including zinc protoporphyrin IX, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG), chromium mesoporphyrin IX (CrMP), tin protoporphyrin IX, and zinc N-methylprotoporphyrin IX. CrMP and ZnBG were found to be the most selective inhibitors of HO activity (i.e., caused the greatest inhibition of HO activity, 89 and 80%, respectively, without inhibition of NOS activity). Based on these results, sGC activity in rat lung cytosol incubated with CrMP or ZnBG (0.15-15 microM) was measured. ZnBG did not affect basal sGC activity but did potentiate S-nitroso-N-acetylpenicillamine (SNAP)-induced sGC activity. CrMP did not affect either basal or SNAP-induced activity. It was concluded that of the five metalloporphyrins studied, CrMP, at a concentration of 5 microM, was a selective inhibitor of HO activity and was the most useful metalloporphyrin for the conditions tested. Thus, CrMP would appear to be a valuable chemical probe in elucidating the physiological role of HO.
A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole-ketones, ... more A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole-ketones, imidazole-dioxolanes, and imidazole-alcohols substituted with halogens in the phenyl ring were synthesized and evaluated as novel inhibitors of heme oxygenase which are structurally distinct from metalloporphyrins. The entire library of compounds was found to be highly active, with the bromine- and iodine-substituted derivatives being the most potent. The imidazole-dioxolanes were all selective for the HO-1 isozyme (inducible) and exhibited substantially lower activity toward the HO-2 isozyme (constitutive). The corresponding imidazole-ketones and imidazole-alcohols showed selectivity toward HO-1 to a lesser degree than the similarly substituted imidazole-dioxolanes.
Drug metabolism and disposition: the biological fate of chemicals
The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bi... more The metabolism of styrene oxide has been studied in the rat and guinea pig, with emphasis upon bivalent sulfur metabolites. Methylthio analogs of phenylethylene glycol, with the methylthio group in both possible positions, were found as urinary metabolites in both species. These compounds were present in more than trace amounts. The excretion of 2-hydroxy-1-methylthio-1-phenylethane amounted to about 7% of the administered dose in the guinea pig, and about 2% in the rat, in o-24 hr urine samples. The positional isomer 1-hydroxy-2-methylthio-1-phenylethane was excreted in lesser amounts in both species. Acidic urinary metabolites derived from glutathione conjugates are species dependent. In this study, the only products observed in the rat were the mercapturic acids expected as a result of reaction of the oxide with glutathione. In the guinea pig, the major bivalent sulfur acids were the corresponding mercaptoacetic acids. Other related metabolites included a mercaptolactic and a mer...
Canadian Journal of Physiology and Pharmacology, Jun 1, 1992
Incubation of glyceryl trinitrate (GTN) with 5% (w/v) rat brain homogenate (RBH) resulted in biot... more Incubation of glyceryl trinitrate (GTN) with 5% (w/v) rat brain homogenate (RBH) resulted in biotransformation of the organic nitrate vasodilator drug to a mixture of glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN). Heating of the RBH at 100 °C for 5 min and (or) pretreatment with 5 mM N-ethylmaleimide at 37 °C for 10 min demonstrated that about two-thirds of the GTN biotransformation activity was due to a sulfhydryl-dependent enzymatic process resulting in the predominant formation of 1,2-GDN, and that the remaining biotransformation activity was due to a sulfhydryl-dependent nonenzymatic process resulting in the selective formation of 1,3-GDN. In a preliminary experiment, nitric oxide formation was observed during the incubation of GTN with RBH under anaerobic conditions. These data support the idea that some of the therapeutic and adverse effects of GTN are mediated through its action in the central nervous system.Key words: glyceryl trinitrate, biotransformation, rat brain homogenate, sulfhydryl-dependent enzyme, nitric oxide formation.
Radioligand binding studies were conducted to test the hypothesis that specific receptor sites fo... more Radioligand binding studies were conducted to test the hypothesis that specific receptor sites for glyceryl trinitrate (GTN) exist in vascular smooth muscle. The radioligand, [3H]GTN (9.98 Ci/mmol), was incubated with whole homogenate or subcellular fractions (10,000 X g pellet, 100,000 X g pellet and 100,000 X g supernatant) of bovine pulmonary vein (BPV) at 4 degrees C, 37 degrees C or room temperature. After incubation for 5, 15, 30 or 60 min, unbound and bound ligand were estimated by physical separation and liquid scintillation spectrometry. Separation of bound from free ligand was accomplished by vacuum filtration, centrifugation, equilibrium dialysis, gel filtration or precipitation. No evidence of specific binding of [3H]GTN to BPV whole homogenate or any subcellular fraction was observed. The viability of the BPV preparations was verified by demonstrating specific binding of [3H]nitrendipine to the 100,000 X g pellet. To ensure that the ligand was present throughout the incubation procedure in sufficient concentration to detect specific binding, the ability of BPV whole homogenate and subcellular fractions to biotransform GTN to glyceryl dinitrate was measured. After 60-min incubation at 37 degrees C, greater than 95% of the GTN added was unaltered; at 90 min, more than 83% of the radioactivity was associated with GTN. Our data are interpreted as not supporting the concept of a GTN receptor in BPV.
We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured r... more We assessed glyceryl trinitrate (GTN) biotransformation and cyclic GMP accumulation in cultured rat lung fibroblasts (RLF), porcine kidney epithelial (PK1), bovine aortic endothelial (BAE) and bovine aortic smooth muscle (BASM) cells. Biotransformation of 0.1 microM GTN was linear over 30 min and the percentage of glyceryl dinitrate (GDN)/10(6) cells for BAE, BASM, RLF and PK1 at 30 min was 3.1, 2.3, 5.8 and 21.7%, respectively. At low GTN concentration (0.01-0.1 microM) there was a highly selective formation of 1,2-GDN, whereas at higher GTN concentration (greater than 1 microM) this selectivity was lost. Cyclic GMP accumulation did not occur in BAE or BASM at any GTN concentration, whereas for RLF and PK1 it was highly correlated to the rate of GDN formation. Upon re-exposure to GTN after treatment of RLF or PK1 cells for 3 hr with 0.1 mM GTN, there was an almost complete loss of the cyclic GMP response, GTN biotransformation was attenuated markedly and the selective formation of 1,2-GDN at low GTN concentration was absent. However, when GTN-treated cells were incubated for 18 hr in GTN-free media, there was a recovery of the cyclic GMP response, GTN biotransformation and selective 1,2-GDN formation toward control values.(ABSTRACT TRUNCATED AT 250 WORDS)
Studies on the physiological role of heme oxygenase (HO) require an inhibitor that will selective... more Studies on the physiological role of heme oxygenase (HO) require an inhibitor that will selectively inhibit HO activity without inhibiting the activity of either nitric oxide synthase (NOS) or soluble guanylyl cyclase (sGC). The objective of this study was to test a series of metalloporphyrins that have previously been shown to inhibit HO activity, for their ability to inhibit HO without inhibiting NOS or sGC activities. Measurement of activity of HO in rat brain microsomes and NOS in rat brain cytosol was made for samples incubated with metalloporphyrins (0.15-50 microM), including zinc protoporphyrin IX, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG), chromium mesoporphyrin IX (CrMP), tin protoporphyrin IX, and zinc N-methylprotoporphyrin IX. CrMP and ZnBG were found to be the most selective inhibitors of HO activity (i.e., caused the greatest inhibition of HO activity, 89 and 80%, respectively, without inhibition of NOS activity). Based on these results, sGC activity in rat lung cytosol incubated with CrMP or ZnBG (0.15-15 microM) was measured. ZnBG did not affect basal sGC activity but did potentiate S-nitroso-N-acetylpenicillamine (SNAP)-induced sGC activity. CrMP did not affect either basal or SNAP-induced activity. It was concluded that of the five metalloporphyrins studied, CrMP, at a concentration of 5 microM, was a selective inhibitor of HO activity and was the most useful metalloporphyrin for the conditions tested. Thus, CrMP would appear to be a valuable chemical probe in elucidating the physiological role of HO.
A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole-ketones, ... more A series of 2-oxy-substituted 1-(1H-imidazol-1-yl)-4-phenylbutanes comprising imidazole-ketones, imidazole-dioxolanes, and imidazole-alcohols substituted with halogens in the phenyl ring were synthesized and evaluated as novel inhibitors of heme oxygenase which are structurally distinct from metalloporphyrins. The entire library of compounds was found to be highly active, with the bromine- and iodine-substituted derivatives being the most potent. The imidazole-dioxolanes were all selective for the HO-1 isozyme (inducible) and exhibited substantially lower activity toward the HO-2 isozyme (constitutive). The corresponding imidazole-ketones and imidazole-alcohols showed selectivity toward HO-1 to a lesser degree than the similarly substituted imidazole-dioxolanes.
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Papers by Kanji Nakatsu